Note: Descriptions are shown in the official language in which they were submitted.
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COMBINATION OF NEAR INFRARED PHOTOIMMUNOTHERAPY TARGETING
CANCER CELLS AND HOST-IMMUNE ACTIVATION
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Application No.
62/655,612 filed April
10, 2018, herein incorporated by reference in its entirety.
FIELD
This disclosure relates to methods of using antibody-IR700 conjugates and in
combination
with one or more immunomodulators to kill cells, such as cancer cells,
following irradiation with
near infrared (NIR) light.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with Government support under project numbers ZO1 ZIA
BC
011513 and ZO1 ZIA BC 010657 by the National Institutes of Health, National
Cancer Institute.
The Government has certain rights in the invention.
BACKGROUND
Although there are several therapies for cancer, there remains a need for
therapies that
effectively kill the tumor cells while not harming non-cancerous cells.
In order to minimize the side effects of conventional cancer therapies,
including surgery,
radiation and chemotherapy, molecularly targeted cancer therapies have been
developed. Among
the existing targeted therapies, monoclonal antibodies (MAb) therapy have the
longest history.
Over 25 therapeutic MAbs have been approved by the Food and Drug
Administration (FDA)
(Waldmann, Nat Med 9:269-277, 2003; Reichert et al., Nat Biotechnol 23:1073-
1078, 2005).
Effective MAb therapy traditionally depends on three mechanisms: antibody-
dependent cellular
cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and receptor
blockade, and
requires multiple high doses of the MAb. MAbs have also been used at lower
doses as vectors to
deliver therapies such as radionuclides (Goldenberg et al., J Clin Oncol 24,
823-834, 2006) or
chemical or biological toxins (Pastan et al., Nat Rev Cancer 6:559-565, 2006).
Ultimately,
however, dose limiting toxicity relates to the biodistribution and catabolism
of the antibody
conjugates.
Conventional photodynamic therapy, which combines a photosensitizing agent
with the
physical energy of non-ionizing light to kill cells, has been less commonly
employed for cancer
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therapy because the currently available non-targeted photosensitizers are also
taken up in normal
tissues, thus, causing side effects, although the excitation light itself is
harmless in the near infrared
(NIR) range. Cancer immunotherapy, which includes the use of immune modulatory
antibodies,
cancer vaccines, and cell-based therapies, has also become a strategy in the
control of cancer (Chen
and Mellman, Immunity 39:1-10, 2013; Childs and Carsten, Nat. Rev. Drug
Discov. 14:487-498,
2015; June et al., Sci. Transl. Med. 7:280ps7, 2015; Melero et al., Nat. Rev.
Cancer 15:457-472,
2015).
Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs
a targeted
monoclonal antibody-photo-absorber conjugate (APC). Following antibody
localization of the
APC to a tumor cell surface antigen, NIR light is used to induce highly
selective cytolysis. NIR-
PIT induces rapid, necrotic cell death that yields innate immune ligands that
activate dendritic cells
(DCs), consistent with immunogenic cell death (ICD). A description of how NIR-
PIT kills tumor
cells is described in Sato et al. (ACS Cent. Sci. 4:1559-69, 2018). Briefly,
following binding of the
antibody-IR700 conjugate to its target, activation by NIR light causes
physical changes in the shape
of antibody-antigen complexes that induce physical stress within the cellular
membrane, leading to
increases in transmembrane water flow that eventually lead to cell bursting
and necrotic cell death.
Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed
to induce durable
regression of established tumors.
SUMMARY OF THE DISCLOSURE
Currently available cancer therapy aims either at directly targeting cancer
cells or activating
host immune system. No currently available cancer therapy achieves both
killing cancer cells and
activating host immune system against cancer cells. Additionally, no current
cancer
immunotherapies successfully produce long-time effective memory T-cells needed
for complete
treatment of cancer without concern about recurrence ¨ a so-called "vaccine"
effect. The methods
disclosed herein can effectively produce long time acting memory T cells that
significantly reduce
or even prevent local or systemic recurrence of cancer.
Provided herein are methods of treating a subject with cancer with a
combination of
antibody-IR700 molecules and NIR-photoimmunotherapy (PIT) with
immunomodulators. In
particular examples, the methods include administering to a subject with
cancer a therapeutically
effective amount of one or more antibody-IR700 molecules, where the antibody
specifically binds
to a cancer cell surface molecule, such as a tumor-specific antigen. The
methods also include
administering to the subject a therapeutically effective amount of one or more
immunomodulators
(such as an immune system activator or an inhibitor of immuno-suppressor
cells), either
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simultaneously or substantially simultaneously with the one or more antibody-
IR700 molecules or
sequentially (for example, within about 0 to 24 hours of one another). The
subject or cancer cells
in the subject (for example, a tumor, or cancer cells in the blood) are then
irradiated at a wavelength
of 660 to 740 nm, such as 660 to 710 nm (for example, 680 nm) at a dose of at
least 1 J/cm2 (such
as at least 50 J/cm2 or at least 100 J/cm2). In some examples, the method can
further include
selecting a subject with cancer having a tumor or cancer that expresses a
cancer cell surface protein
that can specifically bind to the antibody-IR700 molecule.
In some examples, the antibody-IR700 molecule includes an antibody that binds
to one or
more proteins on the cancer cell surface (such as a receptor), wherein the
protein on the cancer cell
surface is not significantly found on non-cancer cells (such as normal healthy
cells) and thus the
antibody will not significantly bind to the non-cancer cells. In one example
the cancer cell surface
protein is a tumor-specific protein, such as CD44, HER1, HER2, or PSMA.
Additional exemplary
tumor-specific proteins and antibodies are provided herein (including in Table
1, below).
In particular embodiments, the immunomodulators include one or more immune
system
activators and/or inhibitors of immuno-suppressor cells, such as an
antagonistic PD-1 antibody,
antagonistic PD-Li antibody, or CD25 antibody-IR700 molecule. In some
examples, the inhibitor
of immuno-suppressor cells inhibits activity and/or kills regulatory T (Treg)
cells. In other
examples, the immune system activator includes one or more interleukins (such
as IL-2 and/or IL-
15). The immunomodulator may, in some examples, increase production of memory
T cells
specific for one or more proteins expressed by the cancer cells.
Also provided are methods of producing memory T cells specific for a target
cell. In
particular examples, the methods include administering to a subject a
therapeutically effective
amount of one or more antibody-IR700 molecules, where the antibody
specifically binds to a cell
surface molecule (such as a tumor-specific protein) on the target cell. The
methods also include
administering to the subject a therapeutically effective amount of one or more
immunomodulators
(such as an immune system activator or an inhibitor of immuno-suppressor
cells), either
simultaneously or substantially simultaneously with the antibody-IR700
molecules or sequentially
(for example, within about 0 to 24 hours). The subject or target cells in the
subject are then
irradiated at a wavelength of 660 to 740 nm, such as 660 to 710 nm (for
example, 680 nm) at a dose
of at least 1 J/cm2 (such as at least 50 J/cm2 or at least 100 J/cm2), thereby
producing memory T
cells.
The foregoing and other features of the disclosure will become more apparent
from the
following detailed description of several embodiments, which proceeds with
reference to the
accompanying figures.
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BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1E are a series of panels showing in vitro effects of NIR-PIT with
anti-CD44-
IR700 on MC38-luc cells. FIG. 1A shows expression of CD44 in MC38-luc cells by
FACS. FIG.
1B is a digital image showing differential interference contrast (DIC) and
fluorescence microscopy
images of control and anti-CD44-IR700 treated MC38-luc cells. Necrotic cell
death was observed
upon excitation with NIR light in treated cells. FIG. 1C is a digital image of
bioluminescence
imaging (BLI) of a 10-cm dish showing NIR light dose-dependent luciferase
activity in MC38-luc
cells. FIG. 1D is a graph showing luciferase activity in MC38-luc cells
treated with NIR and with
or without 10 pg/ml CD44-IR700. FIG. 1E is a graph showing percentage of cell
death in MC38-
luc cells treated with NIR with or without 10 pg/ml CD44-IR700, measured with
dead cell count
using propidium iodide (PI) staining. *, P<0.05 vs. untreated control; **,
P<0.01 vs untreated
control by Student t test.
FIGS. 1F and 1G are graphs showing percentage of cell death in (F) LLC cells
or (G)
MOC1 cells treated with NIR with or without 10 pg/ml CD44-IR700, measured with
dead cell
count using propidium iodide (PI) staining. *, P<0.05 vs. untreated control;
**, P<0.01 vs
untreated control by Student t test.
FIGS. 2A-2C Baseline CD44 expression within MOC1, LLC, and MC38-luc tumor
compartments. (A) size matched MOC1 (day 24), LLC (day 10) and MC38-luc (day
10) tumors
were harvested digested into a single cell suspension, and assessed for CD44
expression on
individual cell types via flow cytometry (n = 3/group). Representative dot
plot and gating strategy
of a tumor digest shown. Cell surface phenotype of each cell type shown above
bar graphs.* *p <
0.01, ***p < 0.001, t test with ANOVA. (B) In vivo CD44-IR700 fluorescence
real-time imaging
of tumor bearing mice. Images were obtained of MOC1 (day 18), LLC (day 4) and
MC38-luc (day
4) tumors 24 hours after i.v. injection of CD44-IR700. The fluorescence
intensity of CD44IR-700
was higher in MC38 tumor compared with the other two tumors. (C) Quantitative
analysis of
IR700 intensities in MOC1, LLC and MC38-luc tumors. The fluorescence
intensities were
significantly higher in MC38-luc tumors compared with other tumors (n 10,***p<
0.001 vs
MOC1 and LLC tumor, Tukey's test with ANOVA).
FIGS. 3A-3G are a series of panels showing in vivo effect of a combination
therapy of
cancer targeting PIT (anti-CD44-IR700) and a checkpoint inhibitor (anti-PD1)
for MC38-luc tumor
in a unilateral tumor model. (A) treatment scheme for unilateral tumor/NIR-PIT
and fluorescence
and bioluminescence imaging at the indicated timepoints; (B) In vivo IR700
fluorescence real-time
imaging of tumor-bearing mice in response to NIR-PIT; (C) In vivo BLI of tumor
bearing mice in
response to NIR-PIT. Mice in the PD-1 mAb group also received CD44-IR700 but
were not treated
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with NIR. (D) Quantification of luciferase activity in four treatment groups
(n 10, **p < 0.01 vs
control, Tukey's t test with ANOVA; #p< 0.05 vs PD-1 mAb and NIR-PIT groups,
Tukey's t test
with ANOVA). (E) Resected tumors (Day 10) were stained with H&E and assessed
for necrosis
and leukocyte infiltration. White scale bars = 100 um. Black scale bars = 20
um. (F) Tumor
growth curves (n 10, **p < 0.01 vs control, Tukey's t test with ANOVA; np
<0.01 vs PD-1
mAb and NIR-PIT groups, Tukey's t test with ANOVA) and (G) Kaplan-Meier
survival analysis
following NIR-PIT treatment with and without PD-1 mAb (**p < 0.01 vs control,
Log rank test; np
<0.01 vs PD-1 mAb and NIR-PIT groups, Log rank test).
FIGS. 4A-4D show the in vivo effect of NIR-PIT and PD-1 mAb in mice bearing a
unilateral LLC tumor. (A) NIR-PIT regimen. Bioluminescence and fluorescence
images were
obtained at each time point as indicated. (B) In vivo IR700 fluorescence real-
time imaging of
tumor-bearing mice in response to NIR-PIT alone or in combination with PD-1
mAb. Mice in the
PD-1 mAb group also received CD44-IR700 but were not treated with NIR. (C) LLC
tumor
growth curves following NIR-PIT treatment with and without PD-1 mAb (n i0,
**p< 0.01 vs
control, ##p< 0.01 vs PD-1 mAb and NIR-PIT groups, Tukey's t test with ANOVA).
(D) Kaplan-
Meier survival analysis (n i0, *p< 0.05, **p< 0.01 vs control, ##p< 0.01 vs PD-
1 mAb and NIR-
PIT groups, Log rank test).
FIGS. 5A-5D show the in vivo effect of NIR-PIT and PD-1 mAb in mice bearing a
unilateral MOC1 tumor. (A) NIR-PIT regimen. Bioluminescence and fluorescence
images were
obtained at each time point as indicated. (B) In vivo IR700 fluorescence real-
time imaging of
tumor-bearing mice in response to NIR-PIT alone or in combination with PD-1
mAb. Mice in the
PD-1 mAb group also received CD44-IR700 but were not treated with NIR. (C)
MOC1 tumor
growth curves following NIR-PIT treatment with and without PD-1 (n i0, **p<
0.01 vs control,
Tukey's test with ANOVA). (D) Kaplan-Meier survival analysis (n i0, *p< 0.05,
**p< 0.01 vs
control, Log rank test).
FIGS. 6A-6F. Immune correlative and functional effects of NIR-PIT and PD-1 mAb
in
mice bearing a unilateral MC38-luc tumor. (A) MC38-luc tumors (day 10, n =
5/group) treated
with NIR-PIT with and without PD-1 mAb and controls were harvested, digested
into single-cell
suspensions, and analyzed for tumor infiltrating lymphocytes (TIL)
infiltration via flow cytometry.
Presented as absolute number of infiltrating cells per 1.5 x 104 live cells
analyzed. PD-1 expression
shown as inset (MFI, mean fluorescence intensity). *p <0.05, **p < 0.01, ***p
<0.001, t test with
ANOVA. (B) Multiplex immunofluorescence was used to validate flow cytometric
data.
Representative 400 x images shown. Quantification of infiltrating TIL from 5
high power fields
(HPF) per tumor, n = 3/group. **p <0.01, ***p <0.001, t test with ANOVA. (C)
TIL were
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extracted from tumors treated as above (n = 5/group) via an IL-2 gradient,
enriched via negative
magnetic selection, and stimulated with irradiated splenocytes pulsed with
peptides representing
known MHC class I-restricted epitopes from selected tumor-associated antigens.
IFNy levels
determined by ELISA from supernatants collected 24 hours after stimulation.
Supernatants from
splenocytes (APC) alone, TIL (T) alone, and a MHC-class I-restricted epitope
from ovalbumin
(OVA, SIINFE,KL) used as controls. *p < 0.05, **p < 0.01, ***p < 0.001, t test
with ANOVA. (D)
Flow cytometric analysis of tumor infiltrating dendritic cells (DC) and
macrophages, with
quantification of macrophage polarization based on MHC class II expression.
**p < 0.01, ***p.<
0.001, t test with ANOVA. (E) Flow cytometric analysis of tumor infiltrating
neutrophilic myeloid
cells (PMN-myeloid) and regulatory T-cells (Tregs). *p < 0.05, **p < 0.01, t
test with ANOVA. (F)
Flow cytometric analysis of PD-Li expression on CD45.2-CD31-PDGFR- tumor cells
and
CD45.2 CD31- immune cells. **p < 0.01 compared to control, t test with ANOVA.
N = 5/group.
FIGS. 7A-7E Immune correlative and functional effects of NIR-PIT and PD-1 mAb
in
mice bearing a unilateral LLC tumor. (A) LLC tumors (day 10, n = 5/group)
treated with NIR-PIT
with and without systemic PD-1 mAb and controls were harvested, digested into
single-cell
suspensions, and analyzed for tumor infiltrating lymphocytes (TIL)
infiltration via flow cytometry.
Presented as absolute number of infiltrating cells per 1.5x 104 live cells
analyzed. PD-1 expression
shown as inset (MFI, mean fluorescence intensity). *p <0.05, **p < 0.01, ***p
<0.001, t test with
ANOVA. (B) TIL were extracted from tumors treated as above (n = 5/group) via
an IL-2 gradient,
enriched via negative magnetic selection, and stimulated with irradiated
splenocytes pulsed with
peptides representing known MHC class I-restricted epitopes from selected
tumor-associated
antigens. IFNy levels determined by ELISA from supernatants collected 24 hours
after stimulation.
Supernatants from splenocytes (APC) alone, TIL (T) alone, and a MHC-class I-
restricted epitope
from ovalbumin (OVA, SIINFEKL) used as controls. *p < 0.05, **p < 0.01, t test
with ANOVA.
(C) Flow cytometric analysis of tumor infiltrating dendritic cells (DC) and
macrophages, with
quantification of macrophage polarization based on MHC class II expression.
**p < 0.01, ***p.<
0.001, t test with ANOVA. (D) Flow cytometric analysis of tumor infiltrating
granulocytic myeloid
derived suppressor cells PMN-myeloid and Tregs. **p <0.01, ***p < 0.001, t
test with ANOVA.
(E) Flow cytometric analysis of PD-Li expression on CD45.2-CD31-PDGFR-tumor
cells and
CD45.2+CD31-immune cells. N = 5/group. *p < 0.05, **p < 0.01, ***p <0.001, t
test with
ANOVA.
FIGS. 8A-8E Immune correlative and functional effects of NIR-PIT and PD-1 mAb
in
MOC1 tumor-bearing mice. (A) MOC1 tumors (day 10, n = 5/group) treated with
NIR-PIT with
and without systemic PD-1 mAb and controls were harvested, digested into
single-cell suspensions
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,and analyzed for tumor infiltrating lymphocytes (TIL) infiltration via flow
cytometry. Presented as
absolute number of infiltrating cells per 1.5x 1041ive cells analyzed. PD-1
expression shown as
inset (MFI, mean fluorescence intensity). *p < 0.05, **p < 0.01, t test with
ANOVA. (B) TIL were
extracted from tumors treated as above (n = 5/group) via an IL-2 gradient,
enriched via negative
magnetic selection, and stimulated with irradiated splenocytes pulsed with
peptides representing
known MHC class I-restricted epitopes from selected tumor-associated antigens.
IFNy levels
determined by ELISA from supernatants collected 24 hours after stimulation.
Supernatants from
splenocytes (APC) alone, TIL (T) alone, and a MHC-class I-restricted epitope
from ovalbumin
(OVA, SIINFE,KL) used as controls. **p < 0.01, t test with ANOVA. (C) Flow
cytometric analysis
of tumor infiltrating dendritic cells (DC) and macrophages, with
quantification of macrophage
polarization based on MHC class II expression. *p < 0.05, **p < 0.01,t test
with ANOVA. (D)
Flow cytometric analysis of tumor infiltrating PMN-myeloid and Tregs. (E) Flow
cytometric
analysis of PD-Li expression on CD45.2-CD31-PDGFR-tumor cells and CD45.2+CD31-
immune
cells. N = 5/group.
FIG. 9 Relative tumor associated antigen gene expression. MC38-luc, LLC and
MOC1
cells were processed and assessed for gene expression of p] 5E, Birb5, Twist]
and Trp53by qRT-
PCR using custom primers designed to flank the region encoding the MHC class I-
restricted
epitope(*p < 0.05, **p < 0.01, ***p < 0.001, t test with ANOVA.). Two-
dimensional plot of
relative antigen expression level vs baseline antigen-specific IFNy responses
in TIL for each model
shown on bottom.
FIGS. 10A-10H In vivo effect of NIR-PIT and PD-1 mAb in mice bearing bilateral
MC38-luc tumors. (A) NIR-PIT regimen. Bioluminescence and fluorescence images
were
obtained at each time point as indicated. (B) NIR light was administered to
the right-sided tumor
only in mice bearing bilateral lower flank tumors. The untreated left-sided
tumor was shielded from
NIR light. (C) In vivo IR700 fluorescence real-time imaging of tumor-bearing
mice in response to
NIR-PIT to the right sided tumor only. (D) In vivo BLI of tumor bearing mice
in response to
combination NIR-PIT and PD-1 mAb. (E) Quantification of luciferase activity
from each tumor, in
controls and mice treated with combination NIR-PIT and PD-1 mAb (n = 10, **p <
0.01, Tukey's
test with ANOVA). (F) Resected tumors (Day 10) were stained with H&E and
assessed for necrosis
and leukocyte infiltration. White scale bars = 100 um. Black scale bars = 20
um. (G) Growth
curves of right- and left-sided tumors from controls and mice treated with
combination NIR-PIT
and PD-1 mAb. (H) Kaplan-Meier survival analysis from controls and mice
treated with
combination NIR-PIT and PD-1 mAb (n = 10, **p < 0.01, Tukey's test with ANOVA
for growth
curves; **p < 0.01, Log-rank test for survival).
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FIGS. 11A-11E. Immune correlative and functional effects of NIR-PIT and PD-1
mAb in
mice bearing a bilateral MC38-luc tumors. (A) Bilateral MC38-luc tumors (day
10, n = 5/group)
treated with PD-1 mAb with or without NIR-PIT and bilateral control tumors
were harvested,
digested into single-cell suspensions, and analyzed for tumor infiltrating
lymphocytes (TIL)
infiltration via flow cytometry. Presented as absolute number of infiltrating
cells per 1.5 x 104 live
cells analyzed. PD-1 expression shown as inset (MFI, mean fluorescence
intensity). *p < 0.05,
***p <0.00i, t test with ANOVA. (B) TIL were extracted from tumors treated as
above (n =
5/group) via an IL-2 gradient, enriched via negative magnetic selection, and
stimulated with
irradiated splenocytes pulsed with peptides representing known MHC class I-
restricted epitopes
from selected tumor-associated antigens. IFNy levels determined by ELISA from
supernatants
collected 24 hours after stimulation. Supernatants from splenocytes (APC)
alone, TIL (T) alone,
and a MHC-class I-restricted epitope from ovalbumin (OVA, SIINFEKL) used as
controls. *p.<
0.05, ***p < 0.001, t test with ANOVA. (C) Flow cytometric analysis of tumor
infiltrating
dendritic cells (DC) and macrophages, with quantification of macrophage
polarization based on
MHC class II expression. **p < 0.01, ***p < 0.001, t test with ANOVA. (D) Flow
cytometric
analysis of tumor infiltrating PMN-myeloid and Tregs. *p < 0.05, **p < 0.01, t
test with ANOVA.
(E) Flow cytometric analysis of PD-Li expression on CD45.2-CD31-PDGFR- tumor
cells. N =
5/group.
FIGS. 12A-12H. In vivo effect of NIR-PIT and PD-1 mAb in mice bearing multiple
MC38-luc tumors. (A) NIR-PIT regimen. Bioluminescence and fluorescence images
were obtained
at each time point as indicated. (B) NIR light was administered to the caudal
right-sided tumor only
in mice bearing four tumors. All other tumors were shielded from NIR light.
(C) In vivo IR700
fluorescence real-time imaging of tumor-bearing mice in response to NIR-PIT
treatment to the
caudal right-sided tumor only. (D) In vivo BLI of tumor bearing mice in
response to NIR-PIT
treatment of the caudal right-sided tumor only. (E) Quantification of
luciferase activity in all
tumors from controls and mice treated with combination NIR-PIT and PD-1 mAb.
Only the caudal
right-sided tumor received NIR-PIT treatment (n = 10, **p < 0.01, Tukey's test
with ANOVA). (F)
Resected tumors (Day 10) were stained with H&E and assessed for necrosis and
leukocyte
infiltration. White scale bars = 100 um. Black scale bars = 20 um. (G) Growth
curves from
controls and treated and untreated tumors from mice receiving combination NIR-
PIT and PD-1
mAb. (H) Kaplan-Meier survival analysis (n = 10, **p < 0.01, Tukey's test with
ANOVA for
growth curves; **p < 0.01, Log-rank test for survival).
FIGS. 13A-13C. Resistance to re-challenge with MC38-luc cells following
complete
tumor rejection with combination NIR-PIT and PD-1 mAb treatment. (A) The
regimen of tumor
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re-challenge in mice that completely rejected (CR) tumors with combination
treatment. Tumor was
inoculated on the contralateral side 30 days after first inoculation. Mice
receiving re-inoculation of
MC38-luc cells. (B) Growth curves of control and CR mice challenged with MC38-
luc cells in the
contralateral flank. (C) Kaplan-Meier survival analysis (n = 9, ***p< 0.001,
by Tukey's test with
ANOVA for growth curves, ***p< 0.001, by Log-rank test for survival).
FIGS. 14A-14C. In vivo IR700 fluorescence imaging of MC38-luc, LL/2, and MOC1
tumor after injection of anti-CD25-mAb-IR700. (A) In vivo anti-CD25-mAb-IR700
fluorescence
real-time imaging of tumor-bearing mice. In MC38-luc, LL/2, and MOC1 tumors,
the tumor
showed high fluorescence intensity after antibody-photo-absorber conjugate
(APC) injection and
the intensity gradually increased up to 24 hours after injection, stabilized
and then decreased after
48 hours. (B) Quantitative analysis of mean fluorescence intensity (MFI) in
MC38-luc, LL/2, and
MOC1 tumors (n = 5 in each group). The MFI of IR700 in MC38-luc, LL/2, and
MOC1 tumors
shows high uptake within 24 hours after APC injection whereupon it decreases
after 48 hours. The
overall MFI over time was significantly higher in MC38-luc tumors compared
with MOC1 tumors
at all time points (*p < 0.05, MC38-luc vs. MOC1 tumors, Tukey-Kramer test),
and the MFI at 24
and 48 hours was significantly higher in LL/2 tumors compared with MOC1 tumors
(**p <0.05,
LL/2 vs. MOC1 tumors, Tukey-Kramer test). (C) Quantitative analysis of target-
to-background
ratio (TBR) in MC38-luc, LL/2, and MOC1 tumors (n = 5 in each group). TBR
gradually increased
up to 24 hours after APC injection, followed by decreased TBR after 48 hours.
The TBR at 24
hours after was significantly higher in MC38-luc and LL/2 tumors compared with
MOC1 tumors
(*p <0.05, MC38-luc vs. MOC1 tumors, Tukey-Kramer test), and the TBR at 48
hours after was
higher in LL/2 tumors compared with MOC1 tumors (**p < 0.05, LL/2 vs. MOC1
tumors, Tukey-
Kramer test).
FIGS. 15A-15F. In vivo effect of CD25- and/or CD44-targeted NIR-PIT for MC38-
luc
tumor model. (A) NIR-PIT regimen. Bioluminescence and fluorescence images were
obtained at
each time point as indicated. (B) In vivo IR700 fluorescence real-time imaging
of tumor bearing
mice in response to NIR-PIT. The tumor treated by NIR-PIT showed decreased
IR700 fluorescence
intensity immediately after NIR-PIT. (C) In vivo bioluminescence imaging of
tumor bearing mice
in response to NIR-PIT. Before NIR-PIT, tumors were approximately the same
size and exhibited
similar bioluminescence. The tumor treated by NIR-PIT showed decreased
luciferase activity after
NIR-PIT, whereupon it either gradually increased (regrowth) or disappeared
(cure). (D)
Quantitative analysis of luciferase activity before and after NIR-PIT in tumor
bearing mice.
Luciferase activity in all NIR-PIT treated groups showed significant decreases
2, 3, 4, 5, 6 and 7
days after NIR-PIT compared to the control group (n = 13-14 mice in each
group, *p < 0.05 vs. the
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other groups, Tukey-Kramer test). Luciferase activity in combined CD25- and
CD44-targeted NIR-
PIT showed significant decrease 7 days after NIR-PIT compared to CD44-targeted
NIR-PIT alone
(n = 13-14 mice in each group, **p < 0.05 vs. combined NIR-PIT group, Tukey-
Kramer test). (E)
Tumor growth in all NIR-PIT treated groups was significantly inhibited 2, 5, 7
and 10 days after
NIR-PIT compared to the control group (n = 13-14 mice in each group, *p < 0.05
vs. the other
groups, Tukey-Kramer test). Combined CD25- and CD44-targeted NIR-PIT showed
significant
tumor reduction 7 and 10 days after NIR-PIT compared to CD44-targeted NIR-PIT
alone (n = 13-
14 mice in each group, **p < 0.05 vs. combined NIR-PIT group, Tukey-Kramer
test). (F)
Significantly prolonged survival was observed in all NIR-PIT treated groups
compared to the
control group (n = 13-14 mice in each group, **p < 0.01, Log-rank test).
Combined CD25- and
CD44-targeted NIR-PIT showed significantly prolonged survival compared to CD25-
targeted NIR-
PIT alone and CD44-targeted NIR-PIT alone (n = 13-14 mice in each group, *p <
0.05, **p < 0.01,
Log-rank test).
FIGS. 16A-16D. In vivo effect of CD25- and/or CD44-targeted NIR-PIT in LL/2
tumor
model. (A) NIR-PIT regimen. IR700 fluorescence images were obtained at each
time point as
indicated. (B) In vivo IR700 fluorescence real-time imaging of tumor-bearing
mice in response to
NIR-PIT. The tumor treated by NIR-PIT showed decreased IR700 fluorescence
intensity
immediately after NIR-PIT. (C) Tumor growth in all NIR-PIT treated groups was
significantly
inhibited 5, 7, 10 and 12 days after NIR-PIT compared to the control group (n
= 9-10 mice in each
group, *p < 0.05 vs. the other groups, Tukey-Kramer test). Among all NIR-PIT
treated groups,
combined CD25- and CD44-targeted NIR-PIT showed significant tumor reduction 17
days after
NIR-PIT compared with CD44-targeted NIR-PIT alone (n = 9 mice in each group,
**p < 0.05 vs.
combined NIR-PIT group, Tukey-Kramer test). (D) Significantly prolonged
survival was observed
in all NIR-PIT treated groups compared to the control group (n = 9-10 mice in
each group, **p <
0.01, Log-rank test). Combined CD25- and CD44-targeted NIR-PIT showed
significantly
prolonged survival compared with CD25-targeted NIR-PIT alone and CD44-targeted
NIR-PIT
alone (n = 9 mice in each group, *p < 0.05, **p < 0.01, Log-rank test).
FIGS. 17A-17D. In vivo effect of CD25- and/or CD44-targeted NIR-PIT in the
MOC1
tumor model. (A) NIR-PIT regimen. IR700 fluorescence images were obtained at
each time point
as indicated. (B) In vivo IR700 fluorescence real-time imaging of tumor-
bearing mice in response
to NIR-PIT. The tumor treated by NIR-PIT showed decreased IR700 fluorescence
intensity
immediately after NIR-PIT. (C) Tumor growth in all NIR-PIT treated groups was
significantly
inhibited 4, 7, 10, 14, 17, 21, 24 and 28 days after NIR-PIT compared to the
control group (n = 9-
10 mice in each group, *p < 0.05 vs. the other groups, Tukey-Kramer test).
Combined CD25- and
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CD44-targeted NIR-PIT showed significant tumor reduction 28 days after NIR-PIT
compared with
CD44-targeted NIR-PIT alone (n = 9-10 mice in each group, **p < 0.05 vs.
combined NIR-PIT
group, Tukey-Kramer test). (D) Significantly prolonged survival was observed
in all NIR-PIT
treated groups compared to the control group (n = 9-10 mice in each group, **p
< 0.01, Log-rank
test). Combined CD25- and CD44-targeted NIR-PIT showed significantly prolonged
survival
compared with CD44-targeted NIR-PIT alone (n = 9-10 mice in each group, **p <
0.01, Log-rank
test).
FIG. 18. Scheme explaining the proposed mechanism of combined CD25- and CD44-
targeted NIR-PIT-induced immunotherapy. Treg cells limit anti-tumor immunity
through
suppression of effector T cells and NK cells by inhibitory cytokines and
cytolysis, as well as by
metabolic disruption with IL-2 consumption, and by modulation of dendritic
cell (DC) maturation
or function. Combined CD25- and CD44-targeted NIR-PIT induces immunogenic cell
death in
CD44+ tumors and selectively depletes Treg cells highly expressing CD25.
First, during the
process of immunogenic cell death, exposure of surface calreticulin, heat
shock protein (Hsp)70/90
and release of ATP and high mobility group box 1 (HMGB1) from dying tumor
cells induce DC
maturation. Second, Treg cell depletion induces activation and expansion of
effector T cells and
NK cells and simultaneously, differentiation into tumor-specific T cells.
Taken together, this
combined NIR-PIT results in effective tumor killing and promotion of long-
lasting anti-tumor
immunity.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Unless otherwise explained, all technical and scientific terms used herein
have the same
meaning as commonly understood by one of ordinary skill in the art to which a
disclosed invention
belongs. The singular terms "a," "an," and "the" include plural referents
unless context clearly
indicates otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly
indicates otherwise. "Comprising" means "including." Hence "comprising A or B"
means
"including A" or "including B" or "including A and B."
Suitable methods and materials for the practice and/or testing of embodiments
of the
disclosure are described below. Such methods and materials are illustrative
only and are not
intended to be limiting. Other methods and materials similar or equivalent to
those described
herein can be used. For example, conventional methods well known in the art to
which a disclosed
invention pertains are described in various general and more specific
references, including, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold
Spring Harbor
Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory
Manual, 3d ed., Cold
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Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular
Biology, Greene
Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short
Protocols in
Molecular Biology: A Compendium of Methods from Current Protocols in Molecular
Biology, 4th
ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor
Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory
Manual, Cold
Spring Harbor Laboratory Press, 1999.
The sequences associated with all GenBank Accession numbers referenced herein
are
incorporated by reference for the sequence available on April 10, 2018.
In order to facilitate review of the various embodiments of the disclosure,
the following
explanations of specific terms are provided:
Administration: To provide or give a subject an agent, such as an antibody-
IR700
molecule and/or an immunomodulator, by any effective route. Exemplary routes
of administration
include, but are not limited to, topical, systemic or local injection (such as
subcutaneous,
intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous),
oral, ocular, sublingual,
rectal, transdermal, intranasal, vaginal, and inhalation routes.
Antibody: A polypeptide ligand comprising at least a light chain or heavy
chain
immunoglobulin variable region which specifically recognizes and binds an
epitope of an antigen,
such as a tumor-specific protein. Antibodies are composed of a heavy and a
light chain, each of
which has a variable region, termed the variable heavy (VH) region and the
variable light (VL)
region. Together, the VH region and the VL region are responsible for binding
the antigen
recognized by the antibody.
Antibodies, such as those in an antibody-IR700 molecule, include intact
immunoglobulins
and the variants and portions of antibodies, such as Fab fragments, Fab
fragments, F(ab)'2
fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv
proteins ("dsFv"). A scFv
protein is a fusion protein in which a light chain variable region of an
immunoglobulin and a heavy
chain variable region of an immunoglobulin are bound by a linker, while in
dsFvs, the chains have
been mutated to introduce a disulfide bond to stabilize the association of the
chains. The term also
includes genetically engineered forms such as chimeric antibodies (for
example, humanized murine
antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See
also, Pierce Catalog
and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J.,
Immunology, 3' Ed., W.
H. Freeman & Co., New York, 1997
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light
(L) chains
interconnected by disulfide bonds. There are two types of light chain, lambda
(X) and kappa (k).
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There are five main heavy chain classes (or isotypes) which determine the
functional activity of an
antibody molecule: IgM, IgD, IgG, IgA and IgE.
Each heavy and light chain contains a constant region and a variable region,
(the regions are
also known as "domains"). In combination, the heavy and the light chain
variable regions
specifically bind the antigen. Light and heavy chain variable regions contain
a "framework" region
interrupted by three hypervariable regions, also called "complementarity-
determining regions" or
"CDRs." The extent of the framework region and CDRs have been defined (see,
Kabat et al.,
Sequences of Proteins of Immunological Interest, U.S. Department of Health and
Human Services,
1991, which is hereby incorporated by reference). The Kabat database is now
maintained online.
The sequences of the framework regions of different light or heavy chains are
relatively conserved
within a species, such as humans. The framework region of an antibody, that is
the combined
framework regions of the constituent light and heavy chains, serves to
position and align the CDRs
in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen.
The CDRs of
.. each chain are typically referred to as CDR1, CDR2, and CDR3, numbered
sequentially starting
from the N-terminus, and are also typically identified by the chain in which
the particular CDR is
located. Thus, a VH CDR3 is located in the variable domain of the heavy chain
of the antibody in
which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of
the light chain of
the antibody in which it is found. Antibodies with different specificities
(i.e. different combining
sites for different antigens) have different CDRs. Although it is the CDRs
that vary from antibody
to antibody, only a limited number of amino acid positions within the CDRs are
directly involved
in antigen binding. These positions within the CDRs are called specificity
determining residues
(SDRs).
References to "VH" or "VH" refer to the variable region of an immunoglobulin
heavy chain,
including that of an Fv, scFv, dsFy or Fab. References to "VL" or "VL" refer
to the variable region
of an immunoglobulin light chain, including that of an Fv, scFv, dsFy or Fab.
A "monoclonal antibody" (mAb) is an antibody produced by a single clone of B
lymphocytes or by a cell into which the light and heavy chain genes of a
single antibody have been
transfected. Monoclonal antibodies are produced for instance by making hybrid
antibody-forming
cells from a fusion of myeloma cells with immune spleen cells. Monoclonal
antibodies include
humanized monoclonal antibodies. In some examples, the antibody in an antibody-
IR700 molecule
is an mAb, such as a humanized mAb.
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A "chimeric antibody" has framework residues from one species, such as human,
and CDRs
(which generally confer antigen binding) from another species, such as a
murine antibody that
specifically binds mesothelin.
A "humanized" immunoglobulin is an immunoglobulin including a human framework
region and one or more CDRs from a non-human (for example a mouse, rat, or
synthetic)
immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a
"donor," and
the human immunoglobulin providing the framework is termed an "acceptor." In
one embodiment,
all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
Constant
regions need not be present, but if they are, they must be substantially
identical to human
immunoglobulin constant regions, e.g., at least about 85-90%, such as about
95% or more identical.
Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are
substantially
identical to corresponding parts of natural human immunoglobulin sequences. A
"humanized
antibody" is an antibody comprising a humanized light chain and a humanized
heavy chain
immunoglobulin. A humanized antibody binds to the same antigen as the donor
antibody that
provides the CDRs. The acceptor framework of a humanized immunoglobulin or
antibody may
have a limited number of substitutions by amino acids taken from the donor
framework.
Humanized or other monoclonal antibodies can have additional conservative
amino acid
substitutions which have substantially no effect on antigen binding or other
immunoglobulin
functions. Humanized immunoglobulins can be constructed by means of genetic
engineering (see
for example, U.S. Patent No. 5,585,089).
A "human" antibody (also called a "fully human" antibody) is an antibody that
includes
human framework regions and all of the CDRs from a human immunoglobulin. In
one example,
the framework and the CDRs are from the same originating human heavy and/or
light chain amino
acid sequence. However, frameworks from one human antibody can be engineered
to include
CDRs from a different human antibody. All parts of a human immunoglobulin are
substantially
identical to corresponding parts of natural human immunoglobulin sequences.
"Specifically binds" refers to the ability of individual antibodies to
specifically
immunoreact with an antigen, such as a tumor-specific antigen, relative to
binding to unrelated
proteins, such as non-tumor proteins, for example 13-actin. For example, a
HER2-specific binding
agent binds substantially only the HER-2 protein in vitro or in vivo. As used
herein, the term
"tumor-specific binding agent" includes tumor-specific antibodies (and
fragments thereof) and
other agents that bind substantially only to a tumor-specific protein in that
preparation.
The binding is a non-random binding reaction between an antibody molecule and
an
antigenic determinant of the T cell surface molecule. The desired binding
specificity is typically
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determined from the reference point of the ability of the antibody to
differentially bind the T cell
surface molecule and an unrelated antigen, and therefore distinguish between
two different
antigens, particularly where the two antigens have unique epitopes. An
antibody that specifically
binds to a particular epitope is referred to as a "specific antibody."
In some examples, an antibody (such as one in an antibody-IR700 molecule)
specifically
binds to a target (such as a cell surface protein, such as a tumor specific
protein) with a binding
constant that is at least 103 M-1 greater, 104M-1 greater or 10 M-1 greater
than a binding constant
for other molecules in a sample or subject. In some examples, an antibody
(e.g., mAb) or
fragments thereof, has an equilibrium constant (Kd) of 1 nM or less. For
example, an antibody
binds to a target, such as tumor-specific protein with a binding affinity of
at least about 0.1 x 10-8
M, at least about 0.3 x 10-8 M, at least about 0.5 x 10-8 M, at least about
0.75 x 10-8 M, at least
about 1.0 x 10-8 M, at least about 1.3 x 10-8 M at least about 1.5 x 10-8M, or
at least about 2.0 x 10-
8 M. Kd values can, for example, be determined by competitive ELISA (enzyme-
linked
immunosorbent assay) or using a surface-plasmon resonance device such as the
Biacore T100,
which is available from Biacore, Inc., Piscataway, NJ.
Antibody-IR700 molecule or antibody-IR700 conjugate: A molecule that includes
both
an antibody, such as a tumor-specific antibody, conjugated to IR700. In some
examples the
antibody is a humanized antibody (such as a humanized mAb) that specifically
binds to a surface
protein on a cancer cell, such as a tumor-specific antigen.
Antigen (Ag): A compound, composition, or substance that can stimulate the
production of
antibodies or a T cell response in an animal, including compositions (such as
one that includes a
tumor-specific protein) that are injected or absorbed into an animal. An
antigen reacts with the
products of specific humoral or cellular immunity, including those induced by
heterologous
antigens, such as the disclosed antigens. "Epitope" or "antigenic determinant"
refers to the region
of an antigen to which B and/or T cells respond. In one embodiment, T cells
respond to the
epitope, when the epitope is presented in conjunction with an MHC molecule.
Epitopes can be
formed both from contiguous amino acids or noncontiguous amino acids
juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids are
typically retained on
exposure to denaturing solvents whereas epitopes formed by tertiary folding
are typically lost on
.. treatment with denaturing solvents. An epitope typically includes at least
3, and more usually, at
least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
Methods of
determining spatial conformation of epitopes include, for example, x-ray
crystallography and
nuclear magnetic resonance.
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Examples of antigens include, but are not limited to, peptides, lipids,
polysaccharides, and
nucleic acids containing antigenic determinants, such as those recognized by
an immune cell. In
some examples, an antigen includes a tumor-specific protein or peptide (such
as one found on the
surface of a cell, such as a cancer cell) or immunogenic fragment thereof.
Cancer: A malignant tumor characterized by abnormal or uncontrolled cell
growth. Other
features often associated with cancer include metastasis, interference with
the normal functioning
of neighboring cells, release of cytokines or other secretory products at
abnormal levels and
suppression or aggravation of inflammatory or immunological response, invasion
of surrounding or
distant tissues or organs, such as lymph nodes, etc. "Metastatic disease"
refers to cancer cells that
have left the original tumor site and migrate to other parts of the body for
example via the
bloodstream or lymph system. In one example, the cell killed by the disclosed
methods is a cancer
cell.
CD25 (IL-2 receptor alpha chain): (e.g., OMIM 147730) A type I transmembrane
protein present on activated T cells, activated B cells, some thymocytes,
myeloid precursors, and
oligodendrocytes. CD25 has been used as a marker to identify CD4+FoxP3+
regulatory T cells in
mice. CD25is found on the surface of some cancer cells, including B-cell
neoplasms, some acute
nonlymphocytic leukemias, neuroblastomas, mastocytosis and tumor infiltrating
lymphocytes. It
functions as the receptor for HTLV-1 and is consequently expressed on
neoplastic cells in adult T
cell lymphoma/leukemia. Exemplary CD25 sequences can be found on the GenBank
database
(e.g., Accession Nos. CAA44297.1, NP_000408.1, and NP_001295171.1). Exemplary
mAbs
specific for CD25 are daclizumab and basiliximab, which can be attached to
IR700, forming
daclizumab-IR700 or basiliximab-IR700, which can be used in the disclosed
methods to target
CD25-expressing cancer cells, or used as an immunomodulator molecule (e.g., to
reduce tumor-
infiltrating Treg cells within the tumor).
CD44: (e.g., OMIM 107269) A cell-surface glycoprotein involved in cell¨cell
interactions,
cell adhesion and migration. CD44 is found on the surface of some cancer
cells, including cancer
stem cells, head and neck cancer cells, breast cancer cells, and prostate
cancer cells. Exemplary
CD44 sequences can be found on the GenBank database (e.g., Accession Nos.
CAJ18532.1,
ACI46596.1, and AAB20016.1). An exemplary mAb specific for CD44 is
bivatuzumab, which can
be attached to IR700, forming bivatuzumab-IR700, which can be used in the
disclosed methods to
target CD44-expressing cancer cells.
Contacting: Placement in direct physical association, including both a solid
and liquid
form. Contacting can occur in vitro, for example, with isolated cells, such as
tumor cells, or in vivo
by administering to a subject (such as a subject with a tumor, such as
cancer).
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Decrease: To reduce the quality, amount, or strength of something. In one
example, a
therapeutic composition that includes one or more antibody-IR700 molecules
decreases the
viability of cells to which the antibody-IR700 molecule specifically binds,
following irradiation of
the cells with NIR (for example at a wavelength of about 680 nm) at a dose of
at least 1 J/cm2, for
example as compared to the response in the absence of the antibody-IR700
molecule. In some
examples such a decrease is evidenced by the killing of the cells. In some
examples, the decrease
in the viability of cells is at least 20%, at least 50%, at least 75%, or even
at least 90%, relative to
the viability observed with a composition that does not include an antibody-
IR700 molecule. In
other examples, decreases are expressed as a fold change, such as a decrease
in the cell viability by
at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 8-
fold, at least 10-fold, or even
at least 15 or 20-fold, relative to the viability observed with a composition
that does not include an
antibody-IR700 molecule. Such decreases can be measured using the methods
disclosed herein.
Immunomodulator: An immunomodulator is a substance that alters (for example,
increases or decreases) one or more functions of the immune system. In some
examples, an
immunomodulator activates the immune system. In other examples, an
immunomodulator inhibits
activity of (or kills) immuno-suppressor cells.
IR700 (IRDye 700DX): A dye having the following formula:
,
_ ....
.9 =() 4, 4
ki
dr,==
LII(..444a ,s
;
= :$0
= = = Si--),õtta
s "WoJa
Moi
1.R.Dyt. 7001:1X NHS Esttv
Commercially available from LI-COR (Lincoln, NE). Amino-reactive IR700 is a
relatively
hydrophilic dye and can be covalently conjugated with an antiboidy using the
NHS ester of IR700.
IR700 also has more than 5-fold higher extinction coefficient (2.1 X 105 M-lcm-
1 at the absorption
maximum of 689 nm), than conventional photosensitizers such as the
hematoporphyrin derivative
Photofrin (1.2 X 103M-1cm-1 at 630 nm), meta-tetrahydroxyphenylchlorin;
Foscan (2.2 X 104
M-lcm-1 at 652 nm), and mono-L-aspartylchlorin e6; NPe6/Laserphyrin (4.0 X
104 M-lcm-lat 654
nm).
Pharmaceutical composition: A chemical compound or composition capable of
inducing
a desired therapeutic or prophylactic effect when properly administered to a
subject. A
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pharmaceutical composition can include a therapeutic agent, such as one or
more antibody-IR700
molecules and/or one or more immunomodulators. A therapeutic or pharmaceutical
agent is one
that alone or together with an additional compound induces the desired
response (such as inducing
a therapeutic or prophylactic effect when administered to a subject). In a
particular example, a
pharmaceutical composition includes a therapeutically effective amount of at
least one antibody-
IR700 molecule.
Pharmaceutically acceptable vehicles: The pharmaceutically acceptable carriers
(vehicles) useful in this disclosure are conventional. Remington: The Science
and Practice of
Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott,
Williams, & Wilkins,
Philadelphia, PA, 21st Edition (2005), describes compositions and formulations
suitable for
pharmaceutical delivery of one or more therapeutic compounds, such as one or
more antibody-
IR700 molecules and/or one or more immunomodulators.
In general, the nature of the carrier will depend on the particular mode of
administration
being employed. For instance, parenteral formulations usually comprise
injectable fluids that
include pharmaceutically and physiologically acceptable fluids such as water,
physiological saline,
balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional non-toxic
solid carriers can
include, for example, pharmaceutical grades of mannitol, lactose, starch, or
magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical compositions to be
administered can
contain minor amounts of non-toxic auxiliary substances, such as wetting or
emulsifying agents,
preservatives, and pH buffering agents and the like, for example sodium
acetate or sorbitan
monolaurate.
Photoimmunotherapy (PIT): A molecularly targeted therapeutic that utilizes a
target-
specific photosensitizer based on a near infrared (NIR) phthalocyanine dye,
IR700, conjugated to
monoclonal antibodies (MAb) targeting cell surface protein. In one example the
cell surface
protein is one found specifically on cancer cells, and thus PIT can be used to
kill such cells. Cell
death occurs when the antibody-IR700 molecule binds to the cells and the cells
are irradiated with
NIR, while cells that do not express the cell surface protein recognized the
antibody-IR700
molecule are not killed in significant numbers.
Programmed death 1 (PD-1): (e.g., OMIM 600244) A type 1 membrane protein on
the
surface of cells that has a role in regulating the immune system's response to
the cells of the human
body by down-regulating the immune system and promoting self-tolerance by
suppressing T cell
inflammatory activity. PD-1 binds to two ligands, PD-Li and PD-L2. Exemplary
PD-1 sequences
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can be found on the GenBank database (e.g., Accession Nos. CAA48113.1,
NP_005009.2, and
NP_001076975.1).
Antibodies that antagonize PD-1 activity can be used as immunomodulators in
the methods
provided herein, for example in combination with a tumor-specific antigen Ab-
IR700 molecule.
Exemplary antagonistic mAbs specific for PD-1 include nivolumab,
pembrolizumab, pidilizumab,
cemiplimab, PDR001, AMP-224, and AMP-514.
Programmed death ligand 1 (PD-L1): (e.g., OMIM 605402) A type 1 membrane
protein
on the surface of cells that suppresses the adaptive arm of immune system
during particular events
such as pregnancy, tissue allografts, autoimmune disease and hepatitis. The
binding of PD-Li to
the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based
on interaction with
phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif
(ITSM) motif.
PD-Li binds to PD-1, found on activated T cells, B cells, and myeloid cells,
to modulate activation
or inhibition. Exemplary PD-Li sequences can be found on the GenBank database
(e.g.,
Accession Nos. ADK70950.1, NP_054862.1, and NP_001156884.1).
Antibodies that antagonize PD-Li activity can be used can be used as
immunomodulators in
the methods provided herein, for example in combination with a tumor-specific
antigen Ab-IR700
molecule. Exemplary antagonistic mAbs specific for PD-Li include atezolizumab,
avelumab,
durvalumab, CK-301, and BMS-936559.
Subject or patient: A term that includes human and non-human mammals. In one
example, the subject is a human or veterinary subject, such as a mouse, rat,
dog, cat, or non-human
primate. In some examples, the subject is a mammal (such as a human) who has
cancer, or is being
treated for cancer.
Therapeutically effective amount: An amount of a composition that alone, or
together
with an additional therapeutic agent(s) (such as a chemotherapeutic agent)
sufficient to achieve a
desired effect in a subject, or in a cell, being treated with the agent. The
effective amount of the
agent (such as an antibody-IR700 molecule, alone or in combination with an
immunomodulator)
can be dependent on several factors, including, but not limited to the subject
or cells being treated,
the particular therapeutic agent, and the manner of administration of the
therapeutic composition.
In one example, a therapeutically effective amount or concentration is one
that is sufficient to
prevent advancement (such as metastasis), delay progression, or to cause
regression of a disease, or
which is capable of reducing symptoms caused by the disease, such as cancer.
In one example, a
therapeutically effective amount or concentration is one that is sufficient to
increase the survival
time of a patient with a tumor.
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In one example, a desired response is to reduce or inhibit one or more
symptoms associated
with cancer. The one or more symptoms do not have to be completely eliminated
for the
composition to be effective. For example, administration of a composition
containing an antibody-
IR700 molecule and a composition containing an immunomodulator (and/or a
single composition
containing both), in combination with irradiation can decrease the size of a
tumor (such as the
volume or weight of a tumor or metastasis of a tumor), for example by at least
20%, at least 50%, at
least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as
compared to the tumor
size in the absence of the treatment. In one particular example, a desired
response is to kill a
population of cells (such as cancer cells) by a desired amount, for example by
killing at least 20%,
at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, or
even at least 100% of the cells, as compared to the cell killing in the
absence of the antibody-IR700
molecule, immunomodulator, and irradiation. In one particular example, a
desired response is to
increase the survival time of a patient with a tumor (or who has had a tumor
recently removed) by a
desired amount, for example increase survival by at least 20%, at least 50%,
at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 100%, at
least 200%, or at least
500%, as compared to the survival time in the absence of the antibody-IR700
molecule,
immunomodulator, and irradiation. In some examples, a desired response is to
increase an amount
of memory T cells in a subject, for example increase by at least 20%, at least
50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least
100%, at least 200%, or at
least 500%, as compared to an amount of memory T cells in the absence of the
antibody-IR700
molecule, immunomodulator, and irradiation. In some examples, a desired
response is to increase
an amount of polyclonal antigen-specific TIC responses against MHC type I-
restricted tumor
specific antigens, in a subject, for example increase by at least 20%, at
least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least
100%, at least 200%, or at
least 500%, as compared to an amount of polyclonal antigen-specific TIC
responses against MHC
type I-restricted tumor specific antigens in the absence of the antibody-IR700
molecule,
immunomodulator, and irradiation. In some examples, a desired response is to
decrease an amount
of Tregs (such as FOXP3 CD25 CD4+ Treg cells), in a targeted tumor, for
example decrease by at
least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, at least
.. 98%, at least 100%, as compared to an amount of Tregs in the targeted tumor
in the absence of the
antibody-IR700 molecule, immunomodulator, and irradiation. In some examples,
combinations of
these effects are archived by the disclosed methods.
The effective amount of an agent that includes one or more of the disclosed
antibody-IR700
molecules (alone or in combination with one or more immunomodulators) that is
administered to a
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human or veterinary subject will vary depending upon a number of factors
associated with that
subject, for example the overall health of the subject. An effective amount of
an agent can be
determined by varying the dosage of the composition(s) and measuring the
resulting therapeutic
response, such as the regression of a tumor. Effective amounts also can be
determined through
various in vitro, in vivo or in situ immunoassays. The disclosed agents can be
administered in a
single dose, or in several doses, as needed to obtain the desired response.
However, the effective
amount can be dependent on the treatment being applied, the subject being
treated, the severity and
type of the condition being treated, and the manner of administration.
In particular examples, a therapeutically effective dose of an antibody-IR700
molecule is at
least 0.5 milligram per 60 kilogram (mg/kg), at least 5 mg/60 kg, at least 10
mg/60 kg, at least 20
mg/60 kg, at least 30 mg/60 kg, at least 50 mg/60 kg, for example 0.5 to 50
mg/60 kg, such as a
dose of 1 mg/ 60 kg, 2 mg/60 kg, 5 mg/60 kg, 20 mg/60 kg, or 50 mg/60 kg, for
example when
administered iv. In another example, a therapeutically effective dose of an
antibody-IR700
molecule is at least 10 ig/kg, such as at least 100 ig/kg, at least 500 ig/kg,
or at least 500 ig/kg,
for example 10 ig/kg to 1000 ig/kg, such as a dose of 100 ig/kg, 250 jig/kg,
about 500 jig/kg, 750
jig/kg, or 1000 jig/kg, for example when administered intratumorally or ip. In
one example, a
therapeutically effective dose is at least 11.1g/ml, such as at least
5001.1g/ml, such as between 20
1.1g/m1 to 1001.1g/ml, such as 101.1g/ml, 201.1g/ml, 301.1g/ml, 401.1g/ml,
501.1g/ml, 601.1g/ml, 70
1.1g/ml, 801.1g/ml, 901.1g/m1 or 1001.1g/m1 administered in topical solution.
However, one skilled in
.. the art will recognize that higher or lower dosages also could be used, for
example depending on
the particular antibody-IR700 molecule. In particular examples, such daily
dosages are
administered in one or more divided doses (such as 2, 3, or 4 doses) or in a
single formulation. The
disclosed antibody-IR700 molecules can be administered alone, in the presence
of a
pharmaceutically acceptable carrier, in the presence of other therapeutic
agents (such as other anti-
neoplastic agents).
Generally a suitable dose of irradiation following administration of the one
or more
antibody-IR700 molecules and one or more immunomodulators is at least 1 J/cm2
at a wavelength
of 660-740 nm, for example, at least 10 J/cm2 at a wavelength of 660-740 nm,
at least 50 J/cm2 at a
wavelength of 660-740 nm, or at least 100 J/cm2 at a wavelength of 660-740 nm,
for example 1 to
.. 500 J/cm2 at a wavelength of 660-740 nm. In some examples the wavelength is
660 ¨ 710 nm. In
specific examples, a suitable dose of irradiation following administration of
the antibody-IR700
molecule is at least 1.0 J/cm2 at a wavelength of 680 nm for example, at least
10 J/cm2 at a
wavelength of 680 nm, at least 50 J/cm2 at a wavelength of 680 nm, or at least
100 J/cm2 at a
wavelength of 680 nm, for example 1 to 500 J/cm2 at a wavelength of 680 nm. In
particular
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examples, multiple irradiations are performed (such as at least 2, at least 3,
or at least 4 irradiations,
such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 separate administrations), following
administration of the
antibody-IR700 molecule and/or the immunomodulator.
Treating: A term when used to refer to the treatment of a cell or tissue with
a therapeutic
agent, includes contacting or incubating one or more agents (such as one or
more antibody-IR700
molecules and one or more immunomodulators) with the cell or tissue and/or
administering one or
more agents to a subject, for example a subject with cancer. A treated cell is
a cell that has been
contacted with a desired composition in an amount and under conditions
sufficient for the desired
response. In one example, a treated cell is a cell that has been exposed to an
antibody-IR700
molecule under conditions sufficient for the antibody to bind to a surface
protein on the cell,
contacted with an immunomodulator, and irradiated with NIR light, until
sufficient cell killing is
achieved. In other examples, a treated subject is a subject that has been
administered one or more
antibody-IR700 molecules under conditions sufficient for the antibody to bind
to a surface protein
on the cell, administered one or more immunomodulators, and irradiated with
NIR light, until
sufficient cell killing is achieved.
Tumor, neoplasia, malignancy or cancer: A neoplasm is an abnormal growth of
tissue or
cells which results from excessive cell division. Neoplastic growth can
produce a tumor. The
amount of a tumor in an individual is the "tumor burden" which can be measured
as the number,
volume, or weight of the tumor. A tumor that does not metastasize is referred
to as "benign." A
tumor that invades the surrounding tissue and/or can metastasize is referred
to as "malignant." A
"non-cancerous tissue" is a tissue from the same organ wherein the malignant
neoplasm formed,
but does not have the characteristic pathology of the neoplasm. Generally,
noncancerous tissue
appears histologically normal. A "normal tissue" is tissue from an organ,
wherein the organ is not
affected by cancer or another disease or disorder of that organ. A "cancer-
free" subject has not
been diagnosed with a cancer of that organ and does not have detectable
cancer.
Tumors include original (primary) tumors, recurrent tumors, and metastases
(secondary)
tumors. A tumor recurrence is the return of a tumor, at the same site as the
original (primary)
tumor, for example, after the tumor has been removed surgically, by drug or
other treatment, or has
otherwise disappeared. A metastasis is the spread of a tumor from one part of
the body to another.
Tumors formed from cells that have spread are called secondary tumors and
contain cells that are
like those in the original (primary) tumor. There can be a recurrence of
either a primary tumor or a
metastasis
Exemplary tumors, such as cancers, that can be treated with the disclosed
methods include
solid tumors, such as breast carcinomas (e.g. lobular and duct carcinomas),
sarcomas, carcinomas
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of the lung (e.g., non-small cell carcinoma, large cell carcinoma, squamous
carcinoma, and
adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, head and
neck cancers
(e.g., adenocarcinoma, squamous cell carcinoma, metastatic squamous, such as
cancers caused by
HPV or Epstein-Barr virus, such as HPV16; can include cancers of the mouth,
tongue,
nasopharynx, throat, hypopharynx, larynx, and trachea), stomach carcinoma,
prostatic
adenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma and
mucinous
cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ
cell tumors,
pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma,
bladder carcinoma
(including, for instance, transitional cell carcinoma, adenocarcinoma, and
squamous carcinoma),
renal cell adenocarcinoma, endometrial carcinomas (including, e.g.,
adenocarcinomas and mixed
Mullerian tumors (carcinosarcomas)), carcinomas of the endocervix, ectocervix,
and vagina (such
as adenocarcinoma and squamous carcinoma of each of same), tumors of the skin
(e.g., squamous
cell carcinoma, basal cell carcinoma, malignant melanoma, skin appendage
tumors, Kaposi
sarcoma, cutaneous lymphoma, skin adnexal tumors and various types of sarcomas
and Merkel cell
carcinoma), esophageal carcinoma, carcinomas of the nasopharynx and oropharynx
(including
squamous carcinoma and adenocarcinomas of same), salivary gland carcinomas,
brain and central
nervous system tumors (including, for example, tumors of glial, neuronal, and
meningeal origin),
tumors of peripheral nerve, soft tissue sarcomas and sarcomas of bone and
cartilage, and lymphatic
tumors (including B-cell and T- cell malignant lymphoma).. In one example, the
tumor is an
adenocarcinoma.
The methods can also be used to treat liquid tumors (e.g., hematological
malignancies),
such as a lymphatic, white blood cell, or other type of leukemia. In a
specific example, the tumor
treated is a tumor of the blood, such as a leukemia (for example acute
lymphoblastic leukemia
(ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML),
chronic
myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic
leukemia (T-
PLL), large granular lymphocytic leukemia, and adult T-cell leukemia),
lymphomas (such as
Hodgkin's lymphoma and non-Hodgkin's lymphoma), and myelomas.
Under conditions sufficient for: A phrase that is used to describe any
environment that
permits the desired activity. In one example, "under conditions sufficient
for" includes
administering an antibody-IR700 molecule to a subject sufficient to allow the
antibody-IR700
molecule to bind to its targeted cell surface protein (such as a tumor-
specific antigen). In particular
examples, the desired activity is killing the cells to which the antibody-
IR700 molecule is bound,
following therapeutic irradiation of the cells.
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Untreated: An untreated cell is a cell that has not been contacted with a
therapeutic agent,
such as an antibody-IR700 molecule, and immunomodulator, and/or irradiation.
In an example, an
untreated cell is a cell that receives the vehicle in which the therapeutic
agent(s) was delivered.
Similarly, an untreated subject is a subject that has not been administered a
therapeutic agent, such
as an antibody-IR700 molecule, and immunomodulator, and/or irradiation. In an
example, an
untreated subject is a subject that receives the vehicle in which the
therapeutic agent(s) was
delivered.
Disclosure of certain specific examples is not meant to exclude other
embodiments. In
.. addition, any treatments described herein are not necessarily exclusive of
other treatment, but can
be combined with other bioactive agents or treatment modalities.
Overview of Technology
Near infrared photoimmunotherapy (NIR-PIT) is a highly-selective cancer
treatment that
.. induces necrotic/immunogenic cell death, utilizing a monoclonal antibody
(mAb) conjugated to a
photo-absorber IR700DX and NIR light. CD44 is associated with resistance to
cancer treatment,
but NIR-PIT employing an anti-CD44-mAb-IR700 conjugate is shown herein to
inhibit cell growth
and prolong survival in multiple tumor types. CD44 mAb-IR700 NIR-PIT targets a
cancer antigen
and initiates necrotic/immunogenic cell death, unlike apoptotic cell death
that most other cancer
therapies induce. Additional treatment with an immunomodulator (such as an
immune checkpoint
inhibitor, for example, an anti-PD1 antibody) synergized the anti-cancer
effects of the anti-CD44-
mAb-IR700 conjugate.
Furthermore, the methods successfully induced reduction of non-PIT treated
distant tumors
(e.g., metastases) and inhibited tumor recurrence upon later challenge with
the same type of tumor
cells. Thus the disclosed methods can also treat recurrences or metastases by
eliciting host
immunity (e.g., in some examples the methods reduce or eliminate tumor
recurrence). PD-1
immune checkpoint blockade (ICB) reversed adaptive immune resistance following
near infrared
photoimmunotherapy to enhance a polyclonal T-cell response and induce
rejection of established
syngeneic tumors in both treated and distant untreated tumors. These
polyclonal responses can also
enhance formation of immunologic memory that suppress recurrence. This work is
the first to
definitively demonstrate development of de novo polyclonal T-cell responses
(e.g., against multiple
tumor associated antigens processed by dendritic cells) following tumor-
targeting cytolytic therapy.
In some examples, the disclosed methods cause selective depletion of Tregs,
increase the number of
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memory T cells (such as tumor antigen specific T cells), increase dendritic
cell tumor infiltration,
or combinations thereof.
In some syngeneic mouse models, FOXP3+CD25 CD4+ Treg cells suppress host anti-
tumor
immunity mediated by inhibiting DC function through the CTLA4 axis or effector
T or NK cell
activation. Increased exposure to tumor antigens in the tumor micro
environment (TME) in the
presence of Treg cells may preferentially activate antigen-specific Treg cells
rather than antigen-
specific effector T cells. To overcome this, cancer and Treg cells were
simultaneously targeted
using combined CD44- and CD25-targeted NIR-PIT, which resulted in superior
anti-tumor effects
and prolonged survival compared to NIR-PIT using either target alone. In
comparison, CD44-
targeted NIR-PIT alone was markedly less effective in all three syngeneic
tumor models
investigated. Although Treg cells mediate tumor immune escape using various
immunosuppressive
mechanisms, CD25-targeted NIR-PIT can disable all of these mechanisms through
selective Treg
cell depletion. These results indicate that the disclosed methods result in
superior in vivo
therapeutic benefits (e.g., tumor growth inhibition and prolonged survival)
over either cancer
.. antigen-targeted NIR-PIT or elimination of immunosuppressive function
alone. This combined
NIR-PIT achieved some complete remissions whereas this was not the case with
either type of
NIR-PIT alone. Thus, the combined NIR-PIT method can result in long-term
survival compared to
conventional cancer antigen-targeted NIR-PIT, likely due to additive effects
of direct tumor killing,
induction of tumor immunogenicity through immunogenic cell death and effective
activation of
host anti-tumor immune cells derived from selective Treg cell depletion by
CD25-targeted NIR-
PIT. These three events, working together may elicit long term tumor responses
in otherwise
resistant tumors. Therefore, combined NIR-PIT with CD25- and CD44-targeted
agents can
eliminate both tumor cells and FOXP3+CD25 CD4+ Treg cells within targeted
tumors. In addition,
combined NIR-PIT simultaneously targeting cancer antigens and
immunosuppressive cells in the
TME may be even more highly efficient than one type of NIR PIT alone, which
can be used to
induce tumor vaccination.
The presence of FOXP3+CD25 CD4+ Treg cells hinders development of tumor-
specific
high-avidity effector T cells although low-avidity effector T cells can
function and expand. Treg-
cell depletion enables activation and expansion of tumor-specific high-avidity
T cells from naive T
cell precursors, allowing their differentiation into high-avidity effector T
cells capable of mediating
potent anti-tumor immune responses. When this occurs, tumor vaccination is
possible with
combined CD25- and CD44-targeted NIR-PIT, due to activation of tumor-specific
high-avidity
effector or memory T cells which can lead to long-lasting anti-tumor immunity
(FIG. 18). NIR-PIT
can be repeatedly performed because it causes minimal damage to surrounding
normal adjacent
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cells. Repeated dosing of antibody-photo-absorber conjugates (APCs) and NIR
light can improve
efficacy of NIR-PIT, increasing the frequency of successful vaccination in
targeted tumors.
Based on these observations, provided herein are methods of treating a subject
using NIR-
PIT in combination with immunomodulation, which can locally kill cancer cells
with minimal
damage to surrounding cells or other cells not targeted by the antibody-IR700
molecule, and also
provide effective anti-tumor host immune activation, resulting in highly
effective treatment of
various cancers using the subject's own immune system, both locally and even
in distant metastases
away from the treated site, with minimal side effects. In some examples,
treatment of a single local
site with the disclosed methods permits systemic host immunity against
cancers, leading to rapid
tumor regression at the treated site as well as untreated distant metastatic
lesions, while inducing
minimal side effects.
Methods for Treating Cancer
The present disclosure provides methods for treating a subject with cancer,
such as a subject
with a tumor or a hematological malignancy. The methods include administering
to the subject an
antibody that is conjugated to the dye IR700 (referred to herein as an
antibody-IR700 molecule),
wherein the antibody specifically binds to a cancer (e.g., tumor) cell surface
protein (also referred
to herein as a tumor-specific antigen or protein). The subject is administered
a therapeutically
effective amount of one or more antibody-IR700 molecules (for example in the
presence of a
pharmaceutically acceptable carrier, such as a pharmaceutically and
physiologically acceptable
fluid), under conditions that permit the antibody to specifically bind to the
cancer cell surface
protein. For example, the antibody-IR700 molecule can be present in a
pharmaceutically effective
carrier, such as water, physiological saline, balanced salt solutions (such as
PBS/EDTA), aqueous
dextrose, sesame oil, glycerol, ethanol, combinations thereof, or the like, as
a vehicle. The carrier
and composition can be sterile, and the formulation suits the mode of
administration. In a specific
example, the antibody-IR700 molecule is CD44 antibody-IR700.
The methods also include administering to the subject a therapeutically
effective amount of
one or more immunomodulators, such as one or more immune system activators
and/or one or more
inhibitors of immuno-suppressor cells (for example in the presence of a
pharmaceutically
acceptable carrier, such as a pharmaceutically and physiologically acceptable
fluid). In a specific
example, the immunomodulatory agent is a PD-1 or PD-Li antibody. In another
specific example,
the immunomodulatory agent is CD25 antibody-IR700. In some examples, the one
or more
immunomodulators are administered to the subject concurrently (for example,
simultaneously or
substantially simultaneously) with the one or more antibody-IR700 molecules
that bind to the
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cancer cell surface protein, for example in the same composition, or if
administered as separate
compositions, within about 1 hour of one another (for example, within about 5
minutes, about 10
minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40
minutes, about 50
minutes, or about 60 minutes). In other examples, the one or more antibody-
IR700 molecules that
bind to the cancer cell surface protein and the one or more immunomodulators
are administered to
the subject sequentially (in either order), for example, separated by at least
about 1 hour to one
week (for example, separated by about 2 hours, about 12 hours, about 24 hours,
about 48 hours,
about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days).
After administering the one or more antibody-IR700 molecules, the one or more
antibody-
IR700 molecules are allowed to accumulate in the targeted tumor. The cancer
cells (or the subject
having the cancer) are then irradiated under conditions that permit killing of
the cells, for example
irradiation at a wavelength of 660 to 710 nm at a dose of at least 1 J/cm2. In
one example, there is
at least about 10 minutes, at least about 30 minutes, at least about 1 hour,
at least about 4 hours, at
least about 8 hours, at least about 12 hours, at least about 24 hours, or at
least about 48 hours (such
as about 1 to 4 hours, 30 minutes to 1 hour, 10 minutes to 60 minutes, 30
minutes to 8 hours, 2 to
10 hours, 12 to 24 hours, 18 to 36 hours, or 24 to 48 hours) in between
administering the antibody-
IR700 molecules and the irradiation. In one example, the one or more antibody-
IR700 molecules
are administered (e.g., i.v.) and at least about 10 minutes, at least about 30
minutes, at least about 1
hour, at least about 4 hours, at least about 8 hours, at least about 12 hours,
at least about 24 hours,
or at least about 48 hours (such as about 1 to 4 hours, 30 minutes to 1 hour,
10 minutes to 60
minutes, 30 minutes to 8 hours, 2 to 10 hours, 12 to 24 hours, 18 to 36 hours,
or 24 to 48 hours,
such as about 24 hours) later, the tumor (or the subject) is irradiated. The
one or more
immunomodulators may be administered before or after the one or more antibody-
IR700 molecules
and/or before or after the irradiation. In some examples, the one or more
immunomodulators are
administered before and after irradiation, for example, at least one dose of
immunomodulators prior
to irradiation and at least one dose of immunomodulators after irradiation
(such as 24 hours before
and one or more of 24, 48, 72, 96, or more hours after irradiation). In
additional examples, a dose
of immunomodulators may also be administered on the same day as at least one
irradiation
treatment.
In some examples, multiple doses of one or more of the antibody-IR700
molecule(s),
immunomodulator(s), and irradiation with NIR are administered to the subject,
such as at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least
9, or at least 10 separate doses (or
administrations). In a specific example, the subject is administered at least
one dose of the one or
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more of the antibody-IR700 molecule(s), at least two doses of the one or more
immunomodulator(s), and at least two administrations of NIR irradiation.
The NIR excitation light wavelength allows penetration of at least several
centimeters into
tissues. For example, by using fiber-coupled laser diodes with diffuser tips,
NIR light can be
delivered within several centimeters of otherwise inaccessible tumors located
deep with respect to
the body surface. In addition to treating solid cancers, circulating tumor
cells (including, but not
limited to hematological malignancies) can be targeted since they can be
excited when they traverse
superficial vessels (for example using the NIR LED wearable devices described
herein).
In one example, administering to the subject one or more antibody-IR700
molecules and
one or more immunomodulators, in combination with irradiation, kills target
cells (such as cancer
cells) that express a cell surface protein (such as a tumor-specific antigen)
that specifically binds to
the antibody. For example, the disclosed methods in some examples kill at
least 10%, for example
at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least
95%, at least 98%, or
more of the treated target cells (such as cancer cells expressing the tumor-
specific antigen) relative
to the absence of treatment with of one or more antibody-IR700 molecules and
administration of
one or more immunomodulators, in combination with irradiation.
In one example, administration of one or more antibody-IR700 molecules and
administration of one or more immunomodulators to a subject having a tumor, in
combination with
irradiation, selectively kills the cells that express a cell surface protein
(such as a tumor-specific
antigen) that can specifically bind to the antibody, thereby treating the
tumor. By selective killing
of tumor cells relative to normal cells is meant that the methods are capable
of killing tumor cells
more effectively than normal cells such as, for example, cells not expressing
the cell surface protein
(such as a tumor-specific antigen) that specifically binds to the antibody
administered. For
example, the disclosed methods in some examples decrease the size or volume of
a tumor, slow the
growth of a tumor, decrease or slow recurrence of the tumor, decrease or slow
metastasis of the
tumor (for example by reducing the number of metastases or decreasing the
volume or size of a
metastasis), or combinations thereof. For example, the disclosed methods in
some examples reduce
tumor size (such as weight or volume of a tumor) or number of tumors and/or
reduce metastatic
tumor size or number of metastatic tumors, such as by at least 10%, for
example by at least 20%, at
least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least
98%, or more, relative to
the absence of administration of one or more antibody-IR700 molecules and
administration of one
or more immunomodulators, in combination with irradiation.
In one example, administration of one or more antibody-IR700 molecules and
administration of one or more immunomodulators to a subject having a tumor, in
combination with
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irradiation, decreases Tregs (such as FOXP3 CD25 CD4+ Treg cells). For
example, the disclosed
methods in some examples decrease the number of circulating Tregs by at least
10%, for example
by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at
least 95%, at least 98%, or
more, relative to the absence of administration of one or more antibody-IR700
molecules and
administration of one or more immunomodulators, in combination with
irradiation. In some
examples, the disclosed methods decrease the number of Tregs in a tumor by at
least 10%, for
example by at least 20%, at least 40%, at least 50%, at least 80%, at least
90%, at least 95%, at
least 98%, or more, relative to the absence of administration of one or more
antibody-IR700
molecules and administration of one or more immunomodulators, in combination
with irradiation.
In one example, administration of one or more antibody-IR700 molecules and
administration of one or more immunomodulators to a subject having a tumor, in
combination with
irradiation, increases memory T cells. For example, the disclosed methods in
some examples
increase the number of circulating memory T cells by at least 10%, for example
by at least 20%, at
least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least
98%, at least 100%, at
least 200%, at least 300%, at least 400%, at least 500%, or more, relative to
the absence of
administration of one or more antibody-IR700 molecules and administration of
one or more
immunomodulators, in combination with irradiation.
In some examples, the disclosed methods decrease one or more symptoms
associated with a
tumor, a recurrence, and/or a metastatic tumor. In one example, the disclosed
methods slow the
growth of a tumor, such as by at least 10%, for example by at least 20%, at
least 40%, at least 50%,
at least 80%, at least 90%, or more, relative to the absence of administration
of the antibody-IR700
molecules and one or more immunomodulators, in combination with irradiation.
In one example,
the disclosed methods reduce or eliminates tumor recurrence, such as by at
least 10%, for example
by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at
least 95%, at least 98%, at
.. least 99% or even 100%, relative to the absence of administration of the
antibody-IR700 molecules
and one or more immunomodulators, in combination with irradiation.
In some examples, the disclosed methods can increase a subject's (such as a
subject with a
tumor or who has had a tumor previously removed) survival time, for example
relative to the
absence of administration of one or more antibody-IR700 molecules and one or
more
.. immunomodulators and irradiation, such as an increase of at least 20%, at
least 40%, at least 50%,
at least 80%, at least 90%, or more. For example, the disclosed methods in
some examples increase
a subject's overall survival time and/or progression-free survival time (for
example, lack of
recurrence of the primary tumor or lack of metastasis) by at least 1 months,
at least 2 months, at
least 3 months, at least 6 months, at least 12 months, at least 18 months, at
least 24 months, at least
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36 months, at least 48 months, at least 60 months, or more, relative to
average survival time in the
absence of administration of an antibody-IR700 molecule, one or more
immunomodulators, and
irradiation.
In one example, administration of a composition containing an antibody-IR700
molecule
and administration of one or more immunomodulators (concurrently or
sequentially), in
combination with NIR irradiation of a primary tumor can decrease the size
and/or number of a
distant non-irradiated tumors or tumor metastases (such as the volume of a
distant tumor or
metastasis, weight of a distant tumor or metastasis, number of distant tumors
or metastases, or
combinations thereof), for example by at least 20%, at least 50%, at least
80%, at least 90%, at least
95%, at least 98%, or even at least 100%, as compared to the
volume/weight/number of distant
tumors or metastases in the absence of the antibody-IR700 molecule, the
immunomodulator, and
NIR irradiation of the primary tumor.
In one example, the disclosed methods increase an amount of polyclonal antigen-
specific
TIC responses against MHC type I-restricted tumor specific antigens, in a
subject, for example
increase by at least 20%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least
95%, at least 98%, at least 100%, at least 200%, or at least 500%, as compared
to an amount of
polyclonal antigen-specific TIC responses against MHC type I-restricted tumor
specific antigens in
the absence of the antibody-IR700 molecule, immunomodulator, and irradiation.
In one example, combinations of these effects are achieved with the disclosed
methods.
The disclosed methods can be used to treat fixed tumors in the body as well as
hematological malignancies and/or tumors in the circulation (e.g., leukemia
cells, metastases,
and/or circulating tumor cells). However, circulating cells, by their nature,
cannot be exposed to
light for very long. Thus, if the cell to be killed is one that is circulating
throughout the body, the
methods can be accomplished by using a device that can be worn, or that covers
parts of the body.
For example, such a device can be worn for extended time periods. Everyday
wearable items (e.g.,
wristwatches, jewelry (such as a necklace or bracelet), blankets, clothing
(e.g., underwear, socks,
and shoe inserts) and other everyday wearable items) which incorporate NIR
emitting light emitting
diodes (LEDs) and a battery pack, can be used. Such devices produce light on
the skin underlying
the device over long periods leading to continual exposure of light to
superficial vessels over
prolonged periods. Circulating tumor cells are exposed to the light as they
transit thru the area
underlying the device. As an example, a wristwatch or bracelet version of this
device can include a
series of NIR LEDs with battery power pack to be worn for most of the day.
After administration
of the one or more antibody-IR700 molecules (e.g., intravenously), circulating
cells bind the
antibody-IR700 conjugate and become susceptible to killing by PIT. As these
cells flow within the
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vessels adjacent to the LED present in the everyday wearable item (e.g.,
bracelet or wristwatch),
they would be exposed to NIR light rendering them susceptible to cell killing.
The dose of light
may be adjustable according to diagnosis and cell type.
In some examples, the method further includes monitoring the therapy, such as
killing of
tumor cells. In such examples, the subject is administered the antibody-IR700
conjugate and
immunomodulators and irradiated as described herein. However, a lower dose of
the antibody-
IR700 conjugate and NIR light can be used for monitoring (as cell killing may
not be required, just
monitoring of the therapy). In one example, the amount of antibody-IR700
conjugate administered
for monitoring is at least 2-fold less (such as at least 3-, 4-, 5-, 6-, 7-, 8-
, 9-, or 10-fold less than the
.. therapeutic dose). In one example, the amount of antibody-IR700 conjugate
administered for
monitoring is at least 20% or at least 25% less than the therapeutic dose. In
one example, the
amount of NIR light used for monitoring is at least 1/1000 or at least
1/10,000 of the therapeutic
dose. This permits detection of the cells being treated. For example, by using
such methods, the
size of the tumor and metastases can be monitored.
In some examples, the method is useful during surgery, such as endoscopic
procedures. For
example, after the antibody-IR700 conjugate and the immunomodulator are
administered to the
subject and the cells irradiated as described above, this not only results in
cell killing, but permits a
surgeon or other medical care provider to visualize the margins of a tumor,
and help ensure that
resection of the tumor (such as a tumor of the skin, breast, lung, colon, head
and neck, or prostate)
is complete and that the margins are clear. In some examples, a lower dose of
the antibody-IR700
conjugate can be used for visualization, such as at least 2-fold less (such as
at least 3-, 4-, 5-, 6-, 7-,
8-, 9-, or 10-fold less than the therapeutic dose).
The antibody-IR700 molecules and immunomodulators can be administered locally
or
systemically, for example to subjects having a tumor, such as a cancer, or who
has had a tumor
previously removed (for example via surgery). Although specific examples are
provided, one
skilled in the art will appreciate that alternative methods of administration
of the disclosed
antibody-IR700 molecules and immunomodulators can be used. Such methods may
include for
example, the use of catheters or implantable pumps to provide continuous
infusion over a period of
several hours to several days into the subject in need of treatment.
In one example, the antibody-IR700 molecules and/or immunomodulators are
administered
by parenteral means, including direct injection or infusion into a tumor
(intratumorally). In some
examples, the antibody-IR700 molecules and/or immunomodulators are
administered to the tumor
by applying the antibody-IR700 molecules and/or immunomodulators to the tumor,
for example by
local injection of antibody-IR700 molecules and/or immunomodulators, bathing
the tumor in a
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solution containing the antibody-IR700 molecules and/or immunomodulators, or
by pouring the
antibody-IR700 molecules and/or immunomodulators onto the tumor.
In addition, or alternatively, the disclosed compositions can be administered
systemically,
for example intravenously, intramuscularly, subcutaneously, intradermally,
intraperitoneally,
subcutaneously, or orally, to a subject having a tumor (such as cancer). The
one or more antibody-
IR700 molecules and one or more immunomodulators may be administered by the
same or
different routes. In one example, the antibody-IR700 molecules may be
administered
intratumorally and the immunomodulators may be delivered systemically (for
example,
intravenously or intraperitoneally). In another example, the antibody-IR700
molecule and the
immunomodulator are administered systemically (for example, intravenously or
intraperitoneally).
In one example, the antibody-IR700 molecule is administered intravenously, and
the
immunomodulator intraperitoneally. In one example, the antibody-IR700 molecule
and the
immunomodulator are administered intravenously.
The dosages of the antibody-IR700 molecules and immunomodulators to be
administered to
a subject are not subject to absolute limits, but will depend on the nature of
the composition, its
active ingredients and its potential unwanted side effects (e.g., immune
response against the
antibody), the subject being treated and the type of condition being treated,
and the manner of
administration. Generally the dose will be a therapeutically effective amount,
such as an amount
sufficient to achieve a desired biological effect, for example an amount that
is effective to decrease
the size (e.g., volume and/or weight) of the tumor, or attenuate further
growth of the tumor, or
decrease undesired symptoms of the tumor.
For intravenous administration of antibody-IR700 molecules (including tumor-
specific
antibody-IR700 molecules and immunomodulator antibody-IR700 molecules),
exemplary dosages
for administration to a subject for a single treatment can range from 0.5 to
100 mg/60 kg of body
weight, 1 to 100 mg/60kg of body weight, 1 to 50 mg/60kg of body weight, 1 to
20 mg/60kg of
body weight, for example about 1 or 2 mg/60kg of body weight. In yet another
example, a
therapeutically effective amount of intraperitoneally or intratumorally
administered antibody-IR700
molecules is 10 lig to 5000 ug of antibody-IR700 molecule per 1 kg of body
weight, such as 10
ug/kg to 1000 jig/kg, 10 jig/kg to 500 jig/kg, or 100 jig/kg to 1000 jig/kg.
In one example, the dose
of antibody-IR700 molecule administered to a human patient is at least 50 mg,
such as at least 100
mg, at least 300 mg, at least 500 mg, at least 750 mg, or even 1 g. Similar
amounts of antibodies
that are not conjugated to IR700 (such as immunomodulator antibodies, such as
those specific for
PD-1 or PD-L1) may also be used.
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Treatments with disclosed antibody-IR700 molecules and immunomodulators can be
completed in a single day, or may be done repeatedly on multiple days with the
same or a different
dosage. Repeated treatments may be done on the same day, on successive days,
or every 1-3 days,
every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, or at even
longer intervals.
In some examples, the antibody-IR700 molecules and immunomodulators are
administered on the
same day. In other examples, the antibody-IR700 molecules and immunomodulators
are
administered on different days. In one non-limiting example, the one or more
antibody-IR700
molecules and one or more immunomodulators are administered to the subject on
the same day and
repeated doses of the one or more immunomodulators (at the same or different
dosing level) are
administered to the subject (for example, 1, 2, 3, 4, 5, or more additional
doses of the
immunomodulator) on successive days, or every 1-3 days, every 3-7 days, every
1- 2 weeks, every
2-4 weeks, every 1-2 months, or at even longer intervals. In some examples,
the amount of the
repeated doses of the immunomodulator is reduced compared to the initial dose
(for example,
reduced by 50% in some cases).
In additional embodiments, the methods also include administering to the
subject one or
more additional therapeutic agents. As described in International Patent
Application Publication
No. WO 2013/009475 (incorporated by reference herein in its entirety), there
is about an 8 hour
window following irradiation (for example irradiation at a wavelength of 660
to 710 nm at a dose
of at least 10 J/cm2, at least 20 J/cm2, at least 30 J/cm2, at least 40 J/cm2,
at least 50 J/cm2, at least
70 J/cm2, at least 80 J/cm2 or at least 100 J/cm2, such as at least 10 to 100
J/cm2), during which
uptake of additional agents (e.g., nano-sized agents, such as those about at
least 1 nm in diameter,
at least 10 nm in diameter, at least 100 nm in diameter, or at least 200 nm in
diameter, such as 1 to
500 nm in diameter) by the PIT-treated cells is enhanced. Thus, one or more
additional therapeutic
agents can further be administered to the subject contemporaneously or
sequentially with the PIT.
In one example, the additional therapeutic agents are administered after the
irradiation, for
example, about 0 to 8 hours after irradiating the cell (such as at least 10
minutes, at least 30
minutes, at least 60 minutes, at least 2 hours, at least 3 hours, at least 4,
hours, at least 5 hours, at
least 6 hours, or at least 7 hours after the irradiation, for example no more
than 10 hours, no more
than 9 hours, or no more than 8 hours, such as 1 hour to 10 hours, 1 hour to 9
hours 1 hour to 8
hours, 2 hours to 8 hours, or 4 hours to 8 hours after irradiation). In
another example, the
additional therapeutic agents are administered just before the irradiation
(such as about 10 minutes
to 120 minutes before irradiation, such as 10 minutes to 60 minutes or 10
minutes to 30 minutes
before irradiation). Additional therapeutic agents that can be used are
discussed below.
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In additional embodiments, methods are provided that permit detection or
monitoring of cell
killing in real-time. Such methods are useful for example, to ensure
sufficient amounts of
antibody-IR700 molecules and/or immunomodulators, or sufficient amounts of
irradiation, were
delivered to the cell or tumor to promote cell killing. These methods permit
detection of cell killing
before morphological changes become evident. In one example, the methods
include contacting a
cell having a cell surface protein with a therapeutically effective amount of
one or more antibody-
IR700 molecules and one or more immunomodulators, wherein the antibody
specifically binds to
the cell surface protein (for example, administering the antibody-IR700
molecule(s) and
immunomodulator(s) to a subject); irradiating the cell at a wavelength of 660
to 740 nm and at a
dose of at least 20 J/cm2; and detecting the cell with fluorescence lifetime
imaging about 0 to 48
hours after irradiating the cell (such as at least 1 hour, at least 2 hours,
at least 4 hours, at least 6
hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36
hours, at least 48 hours, or at
least 72 hours after irradiating the cell, for example 1 minute to 30 minutes,
10 minutes to 30
minutes, 10 minutes to 1 hour, 1 hour to 8 hours, 6 hours to 24 hours, or 6
hours to 48 hours after
irradiating the cell), thereby detecting the cell killing in real-time.
Shortening FLT serves as an
indicator of acute membrane damage induced by PIT. Thus, the cell is
irradiated under conditions
sufficient to shorten IR700 FLT by at least 25%, such as at least 40%, at
least 50%, at least 60% or
at least 75%. In one example, the cell is irradiated at a wavelength of 660 nm
to 740 nm (such as
680 nm to 700 nm) and at a dose of at least 20 J/cm2 or at least 30 J/cm2,
such as at least 40 J/cm2
or at least 50 J/cm2 or at least 60 J/cm2, such as 30 to 50 J/cm2.
In some examples, methods of detecting cell killing in real time includes
contacting the cell
with one or more additional therapeutic agents, for example about 0 to 8 hours
after irradiating the
cell. The real-time imaging can occur before or after contacting the cell with
one or more
additional therapeutic agents. For example, if insufficient cell killing
occurs after administration of
the one or more antibody-IR700 molecules and one or more immunomodulators as
determined by
the real-timing imaging, then the cell can be contacted with one or more
additional therapeutic
agents. However, in some examples, the cell is contacted with the antibody-
IR700 molecules and
immunomodulators and additional therapeutic agents prior to detecting the cell
killing in real-time.
Exemplary cells
The target cell can be a cell that is not desired or whose growth is not
desired, such as a
cancer cell (e.g., a tumor cell). The cells can be present in a mammal to be
treated, such as a
subject (for example, a human or veterinary subject) with cancer. Any target
cell can be treated
with the claimed methods. In one example, the target cell expresses a cell
surface protein that is
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not substantially found on the surface of other normal (desired) cells, an
antibody can be selected
that specifically binds to such protein, and an antibody-IR700 molecule
generated for that protein.
In one example, the cell surface protein is a tumor-specific protein (e.g.,
antigen). In one non-
limiting example, the cell surface protein is CD44.
In one example, the tumor cell is a cancer cell, such as a cell in a patient
with cancer.
Exemplary cells that can be killed with the disclosed methods include cells of
the following tumors:
a hematological malignancy such as a leukemia, including acute leukemia (such
as acute
lymphocytic leukemia, acute myelocytic leukemia, and myeloblastic,
promyelocytic,
myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as
chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera,
lymphoma,
Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrdm's
macroglobulinemia, heavy chain disease). In another example the cell is a
solid tumor cell, such as
cells from sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma,
osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's
tumor,
leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian
cancer, prostate cancer, hepatocellular carcinomna, lung cancer, colorectal
cancer, squamous cell
carcinoma, a head and neck cancer (such as head and neck squamous cell
carcinoma), basal cell
carcinoma, adenocarcinoma (for example adenocarcinoma of the pancreas, colon,
ovary, lung,
breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma,
sebaceous gland
carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary
carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, Wilms' tumor,
cervical cancer, testicular tumor, bladder carcinoma, and CNS cancers (such as
a glioma,
astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma,
hemangioblastoma,
acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and
retinoblastoma).
In a specific example, the cell is a lung cancer cell.
In a specific example, the cell is a breast cancer cell.
In a specific example, the cell is a colon cancer cell.
In a specific example, the cell is a head and neck cancer cell.
In a specific example, the cell is a prostate cancer cell.
Exemplary subjects
In some examples the disclosed methods are used to treat a subject who has
cancer or a
subject with a tumor, such as a tumor described herein. In some examples, the
tumor has been
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previously treated, such as surgically or chemically removed, and the
disclosed methods are used
subsequently to kill any remaining undesired tumor cells that may remain in
the patient and/or
reduce recurrence or metastasis of the tumor.
The disclosed methods can be used to treat any mammalian subject (such as a
human or
veterinary subject, such as a dog or cat), such as a human, who has a tumor,
such as a cancer, or has
had such previously removed or treated. Subjects in need of the disclosed
therapies can include
human subjects having cancer, wherein the cancer cells express a tumor-
specific protein on their
surface that can specifically bind to the antibody-IR700 molecule. For
example, the disclosed
methods can be used as initial treatment for cancer either alone, or in
combination with radiation or
other chemotherapy. The disclosed methods can also be used in patients who
have failed previous
radiation or chemotherapy. Thus, in some examples, the subject is one who has
received other
therapies, but those other therapies have not provided a desired therapeutic
response. The disclosed
methods can also be used in patients with localized and/or metastatic cancer
and/or a recurrence of
a primary tumor.
In some examples the method includes selecting a subject that will benefit
from the
disclosed therapies, such as selecting a subject having a tumor that expresses
a cell surface protein
(such as a tumor-specific protein) that can specifically bind to an antibody-
IR700 molecule. For
example, if the subject is determined to have a breast cancer that expresses
HER2, the subject can
be selected to be treated with an anti-HER2-IR700 molecule, such as
Trastuzumab-IR700 and one
or more immunomodulators, and the subject is subsequently irradiated as
described herein.
Exemplary cell surface proteins
In one example, the protein on the cell surface of the target cell to be
killed is not present in
significant amounts on other cells. For example, the cell surface protein can
be a receptor that is
only found on the target cell type.
In a specific example, the cell surface protein is a cancer- or tumor-specific
protein (also
known in the art as a tumor-specific antigen or tumor-associated antigen),
such as members of the
EGF receptor family (e.g., HER1, 2, 3, and 4) and cytokine receptors (e.g.,
CD20, CD25, IL-13R,
CD5, CD52, etc.). Thus, in some examples, the cell surface protein is an
antigen expressed on the
cell membrane of tumor cells. Tumor-specific proteins are proteins that are
unique to cancer cells
or are much more abundant on them, as compared to other cells, such as normal
cells. For example
HER2 is primarily found in breast cancers, while HER1 is primarily found in
adenocarcinomas,
which can be found in many organs, such as the pancreas, breast, prostate and
colon.
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Exemplary tumor-specific proteins that can be found on a target cell (and to
which an
antibody specific for that protein can be used to formulate an antibody-IR700
molecule), include
but are not limited to: any of the various MAGEs (Melanoma-Associated Antigen
E), including
MAGE 1 (e.g., GenBank Accession Nos. M77481 and AAA03229), MAGE 2 (e.g.,
GenBank
Accession Nos. L18920 and AAA17729), MAGE 3 (e.g., GenBank Accession Nos.
U03735 and
AAA17446), MAGE 4 (e.g., GenBank Accession Nos. D32075 and A06841.1), etc.;
any of the
various tyrosinases (e.g., GenBank Accession Nos. U01873 and AAB60319); mutant
ras; mutant
p53 (e.g., GenBank Accession Nos. X54156, CAA38095 and AA494311); p97 melanoma
antigen
(e.g., GenBank Accession Nos. M12154 and AAA59992); human milk fat globule
(HMFG)
associated with breast tumors (e.g., GenBank Accession Nos. S56151 and
AAB19771); any of the
various BAGEs (Human B melanoma-Associated Antigen E), including BAGE1 (e.g.,
GenBank
Accession No. Q13072) and BAGE2 (e.g., GenBank Accession Nos. NM_182482 and
NP_872288), any of the various GAGEs (G antigen), including GAGE1 (e.g.,
GenBank Accession
No. Q13065) or any of GAGE2-6; various gangliosides, CD25 (e.g., GenBank
Accession Nos.
NP 000408.1 and NM_000417.2).
Other tumor-specific antigens include the HPV 16/18 and E6/E7 antigens
associated with
cervical cancers (e.g., GenBank Accession Nos. NC_001526, FJ952142.1,
ADB94605,
ADB94606, and U89349), mucin (MUC 1)-KLH antigen associated with breast
carcinoma (e.g.,
GenBank Accession Nos. J03651 and AAA35756), CEA (carcinoembryonic antigen)
associated
with colorectal cancer (e.g., GenBank Accession Nos. X98311 and CAA66955),
gp100 (e.g.,
GenBank Accession Nos. S73003 and AAC60634) associated with for example
melanoma,
MARTI antigens associated with melanoma (e.g., GenBank Accession No.
NP_005502), cancer
antigen 125 (CA125, also known as mucin 16 or MUC16) associated with ovarian
and other
cancers (e.g., GenBank Accession Nos. NM_024690 and NP_078966); alpha-
fetoprotein (AFP)
associated with liver cancer (e.g., GenBank Accession Nos. NM_001134 and
NP_001125); Lewis
Y antigen associated with colorectal, biliary, breast, small-cell lung, and
other cancers; tumor-
associated glycoprotein 72 (TAG72) associated with adenocarcinomas; and the
PSA antigen
associated with prostate cancer (e.g., GenBank Accession Nos. X14810 and
CAA32915).
Other exemplary tumor-specific proteins include, but are not limited to, PMSA
(prostate
membrane specific antigen; e.g., GenBank Accession Nos. AAA60209 and
AAB81971.1)
associated with solid tumor neovasculature, as well prostate cancer; HER-2
(human epidermal
growth factor receptor 2, e.g., GenBank Accession Nos. M16789.1, M16790.1,
M16791.1,
M16792.1 and AAA58637) associated with breast cancer, ovarian cancer, stomach
cancer and
uterine cancer, HER-1 (e.g., GenBank Accession Nos. NM_005228 and NP_005219)
associated
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with lung cancer, anal cancer, and gliobastoma as well as adenocarcinomas; NY-
ESO-1 (e.g.
GenBank Accession Nos. U87459 and AAB49693) associated with melanoma,
sarcomas, testicular
carcinomas, and other cancers, hTERT (aka telomerase) (e.g., GenBank
Accession. Nos.
NM_198253 and NP_937983 (variant 1), NM_198255 and NP_937986 (variant 2));
proteinase 3
(e.g., GenBank Accession Nos. M29142, M75154, M96839, X55668, NM 00277,
M96628,
X56606, CAA39943 and AAA36342), and Wilms tumor 1 (WT-1, e.g. GenBank
Accession Nos.
NM_000378 and NP_000369 (variant A), NM_024424 and NP_077742 (variant B),
NM_024425
and NP_077743 (variant C), and NM_024426 and NP_077744 (variant D)).
In one example the tumor-specific protein is CD52 (e.g., GenBank Accession.
Nos.
AAH27495.1 and CAI15846.1) associated with chronic lymphocytic leukemia; CD33
(e.g.,
GenBank Accession. Nos. NM_023068 and CAD36509.1) associated with acute
myelogenous
leukemia; and CD20 (e.g., GenBank Accession. Nos. NP_068769 NP_031667)
associated with
Non-Hodgkin lymphoma.
In a specific example, the tumor-specific protein is CD44 (e.g., OMIM 107269,
GenBank
Accession. Nos. ACI46596.1 and NP_000601.3). CD44 is a marker of cancer stem
cells and is
implicated in intercellular adhesion, cell migration, cell spatial
orientation, and promotion of
matrix-derived survival signal. High expression of CD44 on the plasma membrane
of tumors can
be associated with tumor aggressiveness and poor outcome.
Thus, the disclosed methods can be used to treat any cancer that expresses a
tumor-specific
protein.
Exemplary antibody-IR700 molecules
Because cell surface protein sequences are publically available (for example
as described
above), making or purchasing antibodies (or other small molecules that can be
conjugated to
IR700) specific for such proteins can be accomplished. For example, if the
tumor-specific protein
HER2 is selected as a target, antibodies specific for HER2 (such as
Trastuzumab) can be purchased
or generated and attached to the IR700 dye. Other specific examples are
provided in Table 1. In
one example, the antibody is a humanized monoclonal antibody. Antibody-IR700
molecules can
be generated using methods such as those described in WO 2013/009475
(incorporated by
reference herein in its entirety).
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Table 1. Exemplary tumor-specific antigens and antibodies
Tumor-Specific Exemplary Tumors Exemplary Antibody/Small
Antigen Molecules
HER1 Adenocarcinoma (e.g., Cetuximab, panitumumab,
colorectal cancer, head and zalutumumab, nimotuzumab,
neck cancer) matuzumab, necitumumab,
imgatuzumab, 806. Small
molecule inhibitors gefitinib,
erlotinib, and lapatinib can also
be used.
HER2 breast cancer, ovarian Trastuzumab (Herceptin ),
cancer, stomach cancer, pertuzumab (Peri eta ,
uterine cancer Omnitarg )
HER3 Breast, colon, lung, Patritumab, Duligotumab, MM-
ovarian, prostate, and head 121
and neck squamous cell
cancer
CD19 B cell lymphoma, CLL, GBR 401, MEDI-551,
ALL Blinatumomab (Blincyto )
CD20 Non-Hodgkin lymphoma Tositumomab (Bexxar );
Rituximab (Rituxan, Mabthera);
Ibritumomab tiuxetan (Zevalin,
for example in combination
with yttrium-90 or indium-111
therapy); Ofatumumab
(Arzerra ), veltuzumab,
obinutuzumab, ublituximab,
ocaratuzumab
CD22 Non-Hodgkin's Narnatumab, inotuzumab
lymphoma, CLL, hairy cell ozogamicin, moxetumomab
leukemia, ALL, solid pasudotox
tumors
CD25 T-cell lymphoma Daclizumab (Zenapax),
Basiliximab
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Tumor-Specific Exemplary Tumors Exemplary Antibody/Small
Antigen Molecules
CD30 Hodgkin's lymphoma Brentuximab vedotin
(ADCETRISCI), iratumumab
CD33 Acute myelogenous Gemtuzumab (Mylotarg, for
leukemia example in combination with
calicheamicin therapy);
lintuzumab
CD37 CLL, non-Hodgkin Otlertuzumab
lymphoma, mantle cell
lymphoma
CD38 Multiple myeloma Daratumumab
CD40 Multiple myeloma, non- Lucatumumab, dacetuzumab
Hodgkin's or Hodgkin's
lymphoma
CD44 Cancer stem cells, breast, bivatuzumab
prostate, renal, head and RG3756
neck cancer, lymphoma,
leukemia
CD52 chronic lymphocytic Alemtuzumab (Campath)
leukemia
CD56 Small cell lung cancer, Lorvotuzumab mertansine
ovarian cancer
CD70 Renal cell carcinoma Vorsetuzumab mafodotin
CD74 Multiple myeloma Milatuzumab
CD140 Glioblastoma, non-small Tovetumab
cell lung cancer
CAIX Renal cell carcinoma Girentuximab, cG250
CEA colorectal cancer, some Arcitumomab (CEA-scan (Fab
gastric cancers, biliary fragment, approved by FDA),
cancer colo101; Labetuzumab (CEA-
CideCi)
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Tumor-Specific Exemplary Tumors Exemplary Antibody/Small
Antigen Molecules
Cancer antigen 125 ovarian cancer, 0C125 monoclonal antibody
(CA125) mesothelioma, breast
cancer
Alpha-fetoprotein hepatocellular carcinoma "Y-tacatuzumab tetraxetan;
(AFP) ab75705 (available from
Abcam) and other commercially
available AFP antibodies
Cytokeratin Colorectal cancer 99mTc- Votumumab
(HUMASPECTC))
EGFL7 Non-small cell lung cancer, Pars atuzumab
colorectal cancer
EpCAM Epithelial tumors (breast, IGN101, oportuzumab
colon and lung) monatox, tucotuzumab
celmoleukin, adecatumumab
EPHA3 Lung, kidney and colon KB004, II1A4
tumors, melanoma, glioma
and hematological
malignancies
FAP Colon, breast, lung, Sibrotuzumab, F19
pancreas, and head and
neck tumors
Fibronectin Hodgkin's lymphoma Radretumab
Folate-binding Ovarian cancer MOv18 and MORAb-003
protein (farletuzumab)
Folate receptor Ovarian cancer Farletuzumab
alpha
Frizzled receptor Breast, pancreatic, non- Vantictumab
small cell lung cancer
Gangliosides Neuroectodermal tumors 3F8, ch14.18, KW-2871
(e.g., GD2, GD3 and some epithelial tumors
and GM2)
gpA33 Colorectal cancer huA33
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Tumor-Specific Exemplary Tumors Exemplary Antibody/Small
Antigen Molecules
HGF Solid tumors Rilotumumab, ficlatuzumab
IGF1R Glioma, lung, breast, head Cixutumumab, dalotuzumab,
and neck, prostate and figitumumab, ganitumab,
thyroid cancer robatumumab, teprotumumab,
AVE1642, IMC-Al2, MK-
0646, R1507, and CP 751871
IGLF2 Breast cancer; Dusigitumab
Hepatocellular carcinoma;
Solid tumors
IL-6 renal cell cancer, prostate Siltuximab
cancer, Castleman's
disease
Integrin aV133 Tumor vasculature Etaracizumab (ABEGRINCI),
intetumumab
Integrin a5131 Tumor vasculature Volociximab
Lewis Y colorectal cancer, biliary B3 (Humanized), hu3S193,
cancer IgN311
Mesothelin Mesothelioma, pancreatic Amatuximab
cancer
MET Breast, ovarian, and lung AMG 102, METMAB, SCH
cancer 900105
Mucins Breast, colon, lung and Pemtumomab (THERAGYNCI),
ovarian cancer cantuzumab mertansine, 90Y
clivatuzumab tetraxetan,
oregovomab (OVAREXCI)
PDGFR-alpha Soft tissue sarcoma Olaratumab
Phosphatidylserine Breast, pancreatic, Bavituximab
prostate, non-small cell
lung cancer, hepatocellular
carcinoma
PSMA Prostate cancer J591
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Tumor-Specific Exemplary Tumors Exemplary Antibody/Small
Antigen Molecules
RANKL Prostate cancer, bone Denosumab (XGEVACI)
metastases
Scatter factor Non-small cell lung, Onartuzumab
receptor kinase stomach, glioblastoma
SLAMF7 (CD319) Multiple myeloma Elotuzumab
Syndecan 1 Multiple myeloma, breast, Indatuximab ravtansine
bladder cancer
TAG72 adenocarcinomas including B72.3 (FDA-approved
colorectal, pancreatic, monoclonal antibody), CC49
gastric, ovarian, (minretumomab)
endometrial, mammary,
and non-small cell lung
cancer
Tenascin Glioma, breast and prostate 8106
tumors
TRAILR1 Colon, lung and pancreas Mapatumumab (HGS-ETR1)
tumors and hematological
malignancies
TRAILR2 Non-small cell lung cancer, Conatumumab, lexatumumab,
non-Hodgkin's lymphoma, mapatumumab, tigatuzumab,
multiple myeloma HGS-ETR2, CS-1008
Vascular Colorectal cancer Bevacizumab (AvastinCi)
endothelial growth
factor
VEGFR Epithelium-derived solid IM-2C6, CDP791
tumors
VEGFR2 Gastric, non-small cell Ramucirumab (CyramzaTM)
lung, colorectal cancer
Vimentin Brain cancer Pritumumab
Additional antibodies that can be conjugated to IR700 include 3F8, Abagovomab,
Afutuzumab, Alacizumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab,
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Bectumomab, Belimumab, Besilesomab, Capromab pendetide, Catumaxomab,
Citatuzumab
bogatox, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab,
Ertumaxomab,
Galiximab, Glembatumumab vedotin, Igovomab, Imciromab, Lumiliximab,
Mepolizumab,
Metelimumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab
estafenatox,
Nofetumomab merpentan, Pintumomab, Satumomab pendetide, Sonepcizumab,
Taplitumomab
paptox, Tenatumomab, TGN1412, Ticilimumab (tremelimumab), TNX-650, or
Tremelimumab.
In one example, a patient is treated with at least two different antibody-
IR700 molecules
specific for cancer cell surface antigens. In one example, the two different
antibody-IR700
molecules are specific for the same protein (such as HER-2), but are specific
for different epitopes
of the protein (such as epitope 1 and epitope 2 of HER-2). In another example,
the two different
antibody-IR700 molecules are specific for two different proteins or antigens.
For example, anti-
HER1-IR700 and anti-HER2-IR700 could be injected together as a cocktail to
facilitate killing of
cells bearing either HER1 or HER2.
In one specific example, the antibody-IR700 molecule is anti-CD44-IR700, such
as
RG7356-IR700 or bivatuzumab-IR700. RG7356 is a recombinant human antibody of
the IgGl-
kappa isotype that specifically binds to the constant region of the
extracellular domain of the
human cell-surface glycoprotein CD44 that is present on CD44 standard as well
as on all CD44
splice variants. Bivatuzumab is a humanized mAb specific for CD44 v6.
Immunomodulators
Immunomodulators of use in the disclosed methods include agents or
compositions that
activate the immune system and/or inhibit immuno-suppressor cells (also
referred to herein as
suppressor cells). Without being bound by theory, and as shown in FIG. 18,
inhibition of immuno-
suppressor cells and/or activation of immune responses increases tumor cell
killing and also leads
to production of memory T cells, which can provide a "vaccine" effect against
recurrences and/or
distant tumors or metastases.
In some embodiments, the immunomodulator is an inhibitor of immuno-suppressor
cells,
for example, an agent that inhibits or reduces activity of immuno-suppressor
cells. In some cases,
the immunomodulator kills immuno-suppressor cells. In some examples, the
immuno-suppressor
cells are regulatory T (Treg) cells. In some examples, not all of the
suppressor cells are killed in
vivo, as such could lead to development of autoimmunity. Thus, in some
examples, the method
reduces the activity or number of immuno-suppressor cells in an area of
subject, such as in the area
of a tumor or an area that used to have a tumor, by at least 50%, at least
60%, at least 75%, at least
80%, at least 90%, or at least 95%. In some examples, the method reduces the
total number of
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suppressor cells in a subject by at least 50%, at least 60%, at least 75%, at
least 80%, at least 90%,
or at least 95%.
Inhibitors of immuno-suppressor cells include tyrosine kinase inhibitors (such
as sorafenib,
sunitinib, and imatinib), chemotherapeutic agents (such as cyclophosphamide or
interleukin-toxin
.. fusions, for example denileukin difitox (IL2-diphtheria toxin fusion), or
anti-CD25 antibodies (such
as daclizumab or basiliximab) or other antibodies that bind to suppressor cell
surface proteins (such
as those described below). In other examples, inhibitors of immuno-suppressor
cells include
immune checkpoint inhibitors, for example, anti-PD-1 or anti-PD-Li
antagonizing antibodies,
thereby preventing PD-Li from binding to PD-1 (referred to herein as PD-1/PD-
L1 mAb-mediated
immune checkpoint blockade (ICB)). Thus, in some examples, the immunomodulator
is a PD-1 or
PD-Li antagonizing antibody, such as nivolumab, pembrolizumab, atezolizumab,
avelumab,
durvalumab, MPDL3280A, pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-
936559,
BMS935559, MEDI-4736, MPDL-3280A, MGA-271, indoximod, epacadostat, BMS-986016,
MEDI-4736, MEDI-4737, MK-4166, BMS-663513, PF-05082566 (PF-2566), lirilumab,
and MSB-
0010718C. Checkpoint inhibitors also include anti-CTLA-4 antibodies, including
ipilimumab and
tremelimumab. The inhibitor of immuno-suppressor cells can also be a LAG-3 or
B7-H3
antagonist, such as BMS-986016, and MGA271. In some examples, two or more of
the inhibitors
of immuno-suppressor cells can be administered to a subject. In one non-
limiting example, a
subject is administered an anti-PD1 and an anti-LAG-3 antibody.
In some examples, the agent that inhibits or reduces activity of suppressor
cells includes one
or more antibody-IR700 molecules, wherein the antibody specifically binds to a
suppressor cell
surface protein (such as CD25, CD4, C-X-C chemokine receptor type 4 (CXCR4), C-
C chemokine
receptor type 4 (CCR4), cytotoxic T-lymphocyte-associated protein 4 (CTLA4),
glucocorticoid
induced TNF receptor (GITR), 0X40, folate receptor 4 (FR4), CD16, CD56, CD8,
CD122, CD23,
CD163, CD206, CD11b, Gr-1, CD14, interleukin 4 receptor alpha chain (IL-4Ra),
interleukin-1
receptor alpha (IL-1Ra), interleukin-1 decoy receptor, CD103, fibroblast
activation protein (FAP),
CXCR2, CD33, and CD66b) and in some examples does not include a functional Fc
region (e.g.,
consists of one or more F(ab') 2 fragments). The presence of a functional Fc
portion can result in
autoimmune toxicity (such as antibody-dependent cell-mediated cytotoxicity
(ADCC)). The result
of ADCC is that too many suppressor cells may be killed, instead of only those
suppressor cells
exposed to the NIR light. Thus, the Fc portion of the antibody can be mutated
or removed to
substantially decrease its function (such as a reduction of at least 50%, at
least 75% at least 80%, at
least 90%, at least 95%, at least 99%, or 100% of the Fc function as compared
to a non-mutated Fc
region, such as an ability to bind to the Fcy receptor). Methods and
compositions for reducing
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activity of or killing suppressor cells are described in International Patent
Publication No. WO
2017/027247 (incorporated herein by reference in its entirety).
In a non-limiting example, the immunomodulator is a CD25 antibody-IR700
molecule, such
as daclizumab-IR700 or basiliximab-IR700.
In other embodiments, the immunomodulator is an immune system activator. In
some
examples, an immune system activator stimulates (activates) one or more T
cells and/or natural
killer (NK) cells. In one example, the immune system activator includes one or
more interleukins
(IL), such as IL-2, IL-15, IL-7, IL-12, and/or IL-21. In a non-limiting
example, the
immunomodulator includes IL-2 and IL-15. In another example, the immune system
activator
includes one or more agonists to co-stimulatory receptors, such as 4-1BB,
0X40, or GITR. In a
non-limiting example, the immunomodulator includes stimulatory anti-4-1BB,
anti-0X40, and/or
anti-GITR antibodies.
In some examples, one or more (such as 1, 2, 3, 4, 5, or more) doses of the
immunomodulator is administered to the subject. Thus, administering the
immunomodulator can
be completed in a single day, or may be done repeatedly on multiple days with
the same or a
different dosage (such as administering at least 2 different times, 3
different times, 4 different times
5 different times or 10 different times). In some examples, the repeated
administration are the same
dose. In other examples, the repeated administrations are different does (such
as a subsequent dose
that is higher than the preceding dose or a subsequent dose that is lower than
the preceding dose).
.. Repeated administration of the immunomodulator may be done on the same day,
on successive
days, every other day, every 1-3 days, every 3-7 days, every 1- 2 weeks, every
2-4 weeks, every 1-2
months, or at even longer intervals. In some examples, at least one dose of
the immunomodulator
is administered prior to irradiation and at least one dose of the
immunomodulator is administered
after irradiation.
Irradiation
After the subject is administered one or more antibody-IR700 molecules, and
after (or
optionally before) the subject is administered one or more immunomodulators,
the subject (or a
tumor in the subject) is irradiated. As only cells expressing the cell surface
protein will be
recognized by the antibody, only those cells will have sufficient amounts of
the antibody-IR700
molecules bound to it to kill the cells. This decreases the likelihood of
undesired side effects, such
as killing of normal cells, as the irradiation will only kill the cells to
which the antibody-IR700
molecules are bound, not the other cells.
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The subject (for example, a tumor in the subject) is irradiated with a
therapeutic dose of
radiation at a wavelength of 660 ¨ 710 nm, such as 660-700 nm, 680-7000 nm,
670-690 nm, for
example, 680 nm. In particular examples, the cells are irradiated at a dose of
at least 1 J/cm2, such
as at least 10 J/cm2, at least 30 J/cm2, at least 50 J/cm2, at least 100
J/cm2, or at least 500 J/cm2, for
example, 1-1000 J/cm2, 1-500 J/cm2, 30-50 J/cm2, 10-100 J/cm2, or 10-50 J/cm2.
The subject can be irradiated one or more times. Thus, irradiation can be
completed in a
single day, or may be done repeatedly on multiple days with the same or a
different dosage (such as
irradiation at least 2 different times, 3 different times, 4 different times 5
different times or 10
different times). In some examples, the repeated irradiations are the same
dose. In other examples,
the repeated irradiations are different does (such as a subsequent dose that
is higher than the
preceding dose or a subsequent dose that is lower than the preceding dose).
Repeated irradiations
may be done on the same day, on successive days, every other day, every 1-3
days, every 3-7 days,
every 1- 2 weeks, every 2-4 weeks, every 1-2 months, or at even longer
intervals. In one example,
a first irradiation is 50 J/cm2 and a second irradiation is at 100 J/cm2,
where the irradiations are on
consecutive days (for example, about 24 hours apart).
In some examples, the irradiation is provided with a wearable device
incorporating an NIR
LED. In other examples, another type of device that can be used with the
disclosed methods is a
flashlight-like device with NIR LEDs. Such a device can be used for focal
therapy of lesions
during surgery, or incorporated into endoscopes to apply NIR light to body
surfaces after the
administration of one or more PIT agents. Such devices can be used by
physicians or qualified
health personnel to direct treatment to particular targets on the body.
Treatment using wearable NIR LEDs
As described herein, the disclosed methods are highly specific for cancer
cells. However, in
order to kill the cells circulating in the body or present on the skin, the
patient can wear a device
that incorporates an NIR LED. In some examples, the patient uses at least two
devices, for
example an article of clothing or jewelry during the day, and a blanket at
night. .In some example
the patient uses at least two devices at the same time, for example two
articles of clothing. These
devices make it possible to expose the patient to NIR light using portable
everyday articles of
clothing and jewelry so that treatment remains private and does not interfere
with everyday
activities. In some examples, the device can be worn discreetly during the day
for PIT therapy.
Exemplary devices incorporating an NIR LED are disclosed in International
Patent Application
Publication No. WO 2013/009475 (incorporated by reference herein).
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In one example, the patient is administered one or more antibody-IR700
molecules and one
or more immunomodulators, using the methods described herein. The patient then
wears a device
that incorporates an NIR LED, permitting long-term therapy and treatment of
tumor cells that are
present in the blood or lymph or on the skin. In some examples, the dose is at
least at least 1 J/cm2,
at least 10 J/cm2, at least 20 J/cm2, at least 30 J/cm2, at least 40 J/cm2, or
at least 50 J/cm2, such as
20 J/cm2 or 30 J/cm2. In some examples, administration of the antibody-IR700
molecule is
repeated over a period of time (such as bi-weekly or monthly), to ensure
therapeutic levels are
present in the body.
In some examples, the patient wears or uses the device, or combination of
devices, for at
least 1 week, such as at least 2 weeks, at least 4 weeks, at least 8 weeks, at
least 12 weeks, at least 4
months, at least 6 months, or even at least 1 year. In some examples, the
patient wears or uses the
device, or combination of devices, for at least 4 hours a day, such as at
least 12 hours a day, at least
16 hours a day, at least 18 hours a day, or 24 hours a day. It is quite
possible that multiple devices
of a similar "everyday" nature (blankets, bracelets, necklaces, underwear,
socks, shoe inserts) could
be worn by the same patient during the treatment period. At night the patient
can use the NIR LED
blanket or other covering.
Administration of additional therapies
As discussed above, prior to, during, or following administration of one or
more antibody-
IR700 molecules, immunomodulators, and/or irradiation, the subject can receive
one or more other
therapies. In one example, the subject receives one or more treatments to
remove or reduce the
tumor prior to administration of the antibody-IR700 molecules. In other
examples, additional
treatments or therapeutic agents (such as anti-neoplastic agents) can be
administered to the subject
to be treated, for example, after the irradiation, for example, about 0 to 8
hours after irradiating the
cell (such as at least 10 minutes, at least 30 minutes, at least 60 minutes,
at least 2 hours, at least 3
hours, at least 4, hours, at least 5 hours, at least 6 hours, or at least 7
hours after the irradiation, for
example no more than 10 hours, no more than 9 hours, or no more than 8 hours,
such as 1 hour to
10 hours, 1 hour to 9 hours 1 hour to 8 hours, 2 hours to 8 hours, or 4 hours
to 8 hours after
irradiation). In another example, the additional therapeutic agents are
administered just before the
irradiation (such as about 10 minutes to 120 minutes before irradiation, such
as 10 minutes to 60
minutes or 10 minutes to 30 minutes before irradiation).
Examples of such therapies that can be used in combination with the disclosed
methods,
which enhance accessibility of the tumor to additional therapeutic agents for
about 8 hours after the
PIT, include but are not limited to, surgical treatment for removal or
reduction of the tumor (such
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as surgical resection, cryotherapy, or chemoembolization), as well as anti-
tumor pharmaceutical
treatments which can include radiotherapeutic agents, anti-neoplastic
chemotherapeutic agents,
antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other
agents. In some
examples, the additional therapeutic agent is conjugated to a nanoparticle.
Particular examples of
additional therapeutic agents that can be used include microtubule binding
agents, DNA
intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/or RNA
transcription inhibitors,
antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents
(which are administered
at a therapeutically effective amount) and treatments can be used alone or in
combination. Methods
and therapeutic dosages of such agents are known to those skilled in the art,
and can be determined
by a skilled clinician.
"Microtubule binding agent" refers to an agent that interacts with tubulin to
stabilize or
destabilize microtubule formation thereby inhibiting cell division. Examples
of microtubule
binding agents that can be used in conjunction with the disclosed methods
include, without
limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine
(navelbine), the epothilones,
.. colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin.
Analogs and derivatives of
such compounds also can be used. For example, suitable epothilones and
epothilone analogs are
described in International Publication No. WO 2004/018478. Taxoids, such as
paclitaxel and
docetaxel, as well as the analogs of paclitaxel taught by U.S. Patent Nos.
6,610,860; 5,530,020; and
5,912,264 can be used.
The following classes of compounds can be used with the methods disclosed
herein:
suitable DNA and/or RNA transcription regulators, including, without
limitation, actinomycin D,
daunorubicin, doxorubicin and derivatives and analogs thereof also are
suitable for use in
combination with the disclosed therapies. DNA intercalators and cross-linking
agents that can be
administered to a subject include, without limitation, cisplatin, carboplatin,
oxaliplatin, mitomycins,
such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives
and analogs
thereof. DNA synthesis inhibitors suitable for use as therapeutic agents
include, without limitation,
methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil and analogs thereof.
Examples of suitable
enzyme inhibitors include, without limitation, camptothecin, etoposide,
formestane, trichostatin and
derivatives and analogs thereof. Suitable compounds that affect gene
regulation include agents that
result in increased or decreased expression of one or more genes, such as
raloxifene, 5-azacytidine,
5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and
derivatives and analogs
thereof. Kinase inhibitors include Gleevec (imatinib), Iressa (gefitinib),
and Tarceva
(erlotinib) that prevent phosphorylation and activation of growth factors.
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Non-limiting examples of anti-angiogenic agents include molecules, such as
proteins,
enzymes, polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors,
and small
molecules that function to reduce or even inhibit blood vessel growth.
Examples of suitable
angiogenesis inhibitors include, without limitation, angiostatin K1-3,
staurosporine, genistein,
fumagillin, medroxyprogesterone, suramin, interferon-alpha, metalloproteinase
inhibitors, platelet
factor 4, somatostatin, thromobospondin, endostatin, thalidomide, and
derivatives and analogs
thereof. For example, in some embodiments the anti-angiogenesis agent is an
antibody that
specifically binds to VEGF (e.g., Avastin, Roche) or a VEGF receptor (e.g., a
VEGFR2 antibody).
In one example the anti-angiogenic agent includes a VEGFR2 antibody, or DMXAA
(also known
as Vadimezan or ASA404; available commercially, e.g., from Sigma Corp., St.
Louis, MO) or
both. The anti-angiogenic agent can be bevacizumab, sunitinib, an anti-
angiogenic tyrosine kinase
inhibitors (TM), such as sunitinib, xitinib and dasatinib. These can be used
individually or in any
combination.
Other therapeutic agents, for example anti-tumor agents, that may or may not
fall under one
or more of the classifications above, also are suitable for administration in
combination with the
disclosed methods. By way of example, such agents include adriamycin,
apigenin, rapamycin,
zebularine, cimetidine, and derivatives and analogs thereof.
In some examples, the subject receiving the therapeutic antibody-IR700
molecule
composition is also administered interleukin-2 (IL-2), for example via
intravenous administration.
In particular examples, IL-2 (Chiron Corp., Emeryville, CA) is administered at
a dose of at least
500,000 IU/kg as an intravenous bolus over a 15 minute period every eight
hours beginning on the
day after administration of the antibody-IR700 molecules and continuing for up
to 5 days. Doses
can be skipped depending on subject tolerance.
Exemplary additional therapeutic agents include anti-neoplastic agents, such
as
chemotherapeutic and anti-angiogenic agents or therapies, such as radiation
therapy. In one
example the agent is a chemotherapy immunosuppressant (such as Rituximab,
steroids) or a
cytokine (such as GM-CSF). Chemotherapeutic agents are known in the art (see
for example,
Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's
Principles of Internal
Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff,
Clinical Oncology 2nd ed.,
2000 Churchill Livingstone, Inc; Baltzer and Berkery. (eds): Oncology Pocket
Guide to
Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer Knobf, and
Durivage (eds):
The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).
Combination
chemotherapy is the administration of more than one agent to treat cancer.
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Exemplary chemotherapeutic agents that can be used with the methods provided
herein
include but are not limited to, carboplatin, cisplatin, paclitaxel, docetaxel,
doxorubicin, epirubicin,
topotecan, irinotecan, gemcitabine, iazofurine, gemcitabine, etoposide,
vinorelbine, tamoxifen,
valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone, Doxil
(liposome
encapsulated doxiorubicine) and vinorelbine. Additional examples of
chemotherapeutic agents that
can be used include alkylating agents, antimetabolites, natural products, or
hormones and their
antagonists. Examples of alkylating agents include nitrogen mustards (such as
mechlorethamine,
cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates
(such as busulfan),
nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or
dacarbazine). Specific
non-limiting examples of alkylating agents are temozolomide and dacarbazine.
Examples of
antimetabolites include folic acid analogs (such as methotrexate), pyrimidine
analogs (such as 5-FU
or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.
Examples of natural
products include vinca alkaloids (such as vinblastine, vincristine, or
vindesine),
epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as
dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes
(such as L-
asparaginase). Examples of miscellaneous agents include platinum coordination
complexes (such
as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas
(such as
hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and
adrenocrotical suppressants
(such as mitotane and aminoglutethimide). Examples of hormones and antagonists
include
adrenocorticosteroids (such as prednisone), progestins (such as
hydroxyprogesterone caproate,
medroxyprogesterone acetate, and magestrol acetate), estrogens (such as
diethylstilbestrol and
ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as
testosterone
proprionate and fluoxymesterone).
Examples of commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-
C,
BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin,
DTIC, 5-
fluoruracil (5-FU), Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate,
Mithramycin,
Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as
docetaxel), Velban,
Vincristine, VP-16, Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar,
CPT-11), Leustatin,
Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda
(Capecitabine), Zevelin and
calcitriol. Non-limiting examples of immunomodulators that can be used include
AS-101 (Wyeth-
Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF
(granulocyte
macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or
Hoffman-LaRoche),
human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans,
La.), SK&F
106528, and TNF (tumor necrosis factor; Genentech).
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In some examples, the additional therapeutic agent is conjugated to (or
otherwise associated
with) a nanoparticle, such as one at least 1 nm in diameter (for example at
least 10 nm in diameter,
at least 30 nm in diameter, at least 100 nm in diameter, at least 200 nm in
diameter, at least 300 nm
in diameter, at least 500 nm in diameter, or at least 750 nm in diameter, such
as 1 nm to 500 nm, 1
nm to 300 nm, 1 nm to 100 nm, 10 nm to 500 nm, or 10 nm to 300 nm in
diameter).
In one example, at least a portion of the tumor (such as a metastatic tumor)
is surgically
removed (for example via surgical resection and/or cryotherapy), irradiated
(for example
administration of radioactive material or energy (such as external beam
therapy) to the tumor site to
help eradicate the tumor or shrink it), chemically treated (for example via
chemoembolization) or
combinations thereof, prior to administration of the disclosed therapies (such
as administration of
antibody-IR700 molecules and/or immunomodulators). For example, a subject
having a metastatic
tumor can have all or part of the tumor surgically excised prior to
administration of the disclosed
therapies. In an example, one or more chemotherapeutic agents are administered
following
treatment with antibody-IR700 molecules, immunomodulators, and irradiation. In
another
particular example, the subject has a metastatic tumor and is administered
radiation therapy,
chemoembolization therapy, or both concurrently with the administration of the
disclosed therapies.
In some examples, the additional therapeutic agent administered is a
monoclonal antibody,
for example, 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab ,
Alemtuzumab,
Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab,
Bavituximab,
Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine,
Blinatumomab,
Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab,
CC49,
Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan,
Conatumumab,
Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab,
Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab
ozogamicin,
Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab,
Imciromab,
Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab,
Lexatumumab,
Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab,
Matuzumab,
Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab
tafenatox,
Naptumomab estafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan,
Ofatumumab,
Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab,
Pertuzumab,
Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab,
Satumomab
pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab tetraxetan, Taplitumomab
paptox,
Tenatumomab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-650,
Trastuzumab,
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Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab,
Zalutumumab, or combinations thereof.
Production of Memory T cells
Also provided are methods of producing memory T cells specific for a target
cell. In
particular examples, the methods include administering to a subject a
therapeutically effective
amount of one or more antibody-IR700 molecules, where the antibody
specifically binds to a target
cell surface molecule, such as a tumor specific antigen (such as those listed
in Table 1). The
methods also include administering to the subject a therapeutically effective
amount of one or more
immunomodulators (such as an immune system activator or an inhibitor of immuno-
suppressor
cells), either simultaneously or substantially simultaneously with the
antibody-IR700 molecules or
sequentially (for example, within about 0 to 24 hours). In a specific example,
the
immunomodulatory agent is a PD-1 or PD-Li antagonistic antibody. In another
specific example,
the immunomodulatory agent is a CD25 antibody-IR700 molecule. The subject or
cells in the
subject are then irradiated at a wavelength of 660 to 740 nm, such as 660 to
710 nm (for example,
680 nm) at a dose of at least 1 J/cm2 (such as at least 50 J/cm2 or at least
100 J/cm2).
Memory T cells may be either CD4+ or CD8+ and usually express CD45RO. Thus, in
some
examples, memory T cells are identified by detecting cells expressing CD45RO.
A number of
subtypes of memory T cells are known. For example, central memory T cells (Tcm
cells) express
CD45RO, C-C chemokine receptor type 7 (CCR7), and L-selectin (CD62L). Central
memory T
cells also have intermediate to high expression of CD44. This memory
subpopulation is commonly
found in the lymph nodes and in the peripheral circulation. Effector memory T
cells (TEm cells and
TEmRA cells) express CD45RO, but lack expression of CCR7 and L-selectin. They
also have
intermediate to high expression of CD44. These memory T cells lack lymph node-
homing
receptors and are thus found in the peripheral circulation and tissues. TEmRA
cells also express
CD45RA. Tissue resident memory T cells (TRm) express integrin ae137. Specific
to TRms are genes
involved in lipid metabolism, being highly active, roughly 20- to 30-fold more
active than in other
types of T-cells. Stem memory (Tscm cells) are CD45R0-, CCR7, CD45RA+, CD62L+
(L-
selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of
CD95,
CXCR3, and LFA-1.
In some examples, the disclosed methods increase memory T cells by at least 1%
(for
example, at least 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 2-fold, 3-
fold, 4-fold, 5-fold, 10-fold, or more) compared to the amount of memory T
cells in a subject who
has not been treated with the disclosed methods. In some examples, total
memory T cells are
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increased, while in other examples, one or more subtypes of memory T cells are
increased
compared to an untreated subject. In other examples, the methods increase
memory T cells for a
specific antigen, such as a tumor-specific antigen, by at least 1% (for
example, at least 2%, 5%,
7%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 2-fold, 3-fold, 4-fold,
5-fold, 10-
fold, or more) compared to the amount of memory T cells in a subject who has
not been treated
with the disclosed methods. In non-limiting examples, the memory T cells
recognize one or more
of pl5E, birc5, twist, and p53 (see Example 3).
The number and/or type of memory T cells can be determined in a sample from a
subject
(such as a treated subject). In some examples, immunoassays and/or genetic
analysis are used to
detect memory T cells in a blood sample from a subject. For example, presence
and/or amount of
one or more memory T cell surface markers can be measured, for example by flow
cytometry. In
another example, tumor infiltrating lymphocytes (TIL) can be obtained from a
treated subject, and
checked for functional reactivity against antigens, such as tumor-specific
antigens. Exemplary
methods are provided in Example 3, below. The number, type, and/or reactivity
profile of memory
T cells can be compared to a control, such as an untreated subject, the
subject prior to treatment,
and/or a reference number (such as an average obtained from a population of
normal (e.g., healthy)
individuals).
Production of polyclonal antigen-specific TIC
Also provided are methods of increasing polyclonal antigen-specific TIC
responses against
MHC type I-restricted tumor specific antigens. In particular examples, the
methods include
administering to a subject a therapeutically effective amount of one or more
antibody-IR700
molecules, where the antibody specifically binds to a target cell surface
molecule, such as a tumor
specific antigen (such as those listed in Table 1). The methods also include
administering to the
subject a therapeutically effective amount of one or more immunomodulators
(such as an immune
system activator or an inhibitor of immuno-suppressor cells), either
simultaneously or substantially
simultaneously with the antibody-IR700 molecules or sequentially (for example,
within about 0 to
24 hours). In a specific example, the immunomodulatory agent is a PD-1 or PD-
Li antagonistic
antibody. In another specific example, the immunomodulatory agent is a CD25
antibody-IR700
molecule. The subject or cells in the subject are then irradiated at a
wavelength of 660 to 740 nm,
such as 660 to 710 nm (for example, 680 nm) at a dose of at least 1 J/cm2
(such as at least 50 J/cm2
or at least 100 J/cm2).
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Example 1
Materials and Methods
This example describes materials and methods used to obtain the results in
Examples 2-9
(see also Nagaya et al., Cancer Immunol. Res. 7:401-13, 2019, herein
incorporated by reference in
its entirety).
Reagents
Water soluble, silica-phthalocyanine derivative, IRDye 700DX NHS ester (IR700)
was
from LI-COR Biosciences (Lincoln, NE, USA). Anti-mouse/human CD44-specific mAb
(clone
IM7) and an anti-mouse PD-1 (CD279) specific mAb (clone RMP1-14) were from
BioXCell (West
Lebanon, NH, USA). All other chemicals were of reagent grade.
Synthesis of IR700-conjugated anti-CD44 mAb
Anti-CD44 mAb (1.0 mg, 6.7 nmol) was incubated with IR700 NHS ester (65.1 pg,
33.3
nmol) in 0.1 M Na2HPO4 (pH 8.6) at room temperature for 1 h and purified with
a Sephadex G25
column (PD-10; GE Healthcare, Piscataway, NJ, USA) . Protein concentration was
determined with
Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL,
USA) by measuring
the absorption at 595 nm with UV-Vis (8453 Value System; Agilent Technologies,
Santa Clara,
CA, USA). IR700 concentration was measured by absorption at 689 nm to confirm
the number of
fluorophore molecules per mAb. CD44-IR700 conjugate synthesis was controlled
so that an
average of two IR700 molecules were bound to each CD44 antibody. Fluorescence
at 700 nM and
molecular weight of CD44-IR700 conjugates was verified using sodium dodecyl
sulfate-
polyacrylamide (4-20% gradient) gel electrophoresis (SDS-PAGE).
Cell culture
MC38 (colon cancer) cells stably expressing luciferase (MC38-luc), LLC (Lewis
lung
carcinoma) cells, and MOC1 (murine oral carcinoma) cells were maintained in
culture as
previously described (Farsaci et al., Cancer Immunol Res. 2014; 2:1090-102;
Hodge et al., Cancer
Biother Radiopharm. 2012; 27:12-22; Judd et al., Cancer Res. 2012; 72:365-74).
Cells were
maintained in culture for no more than 30 passages and routinely tested
negative for mycoplasma.
In vitro NIR-PIT
MC38-luc, LLC or MOC1 cells (2 x 105) were seeded into 12 well plates,
incubated for 24
h, then exposed to media containing 10 lig/mL of CD44-IR700 for 6 h at 37 C.
Cells were
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irradiated with a red light-emitting diode (LED, 690 20 nm wavelength, L690-
66-60; Marubeni
America Co., Santa Clara, CA, USA) at a power density of 50 mW/cm2. Cells were
harvested with
a cell scraper, stained with propidium iodide (PI, 2 lig/mL) at room
temperature for 30 mm, then
analyzed on a BD FACSCalibur (BD Biosciences) using CellQuest software.
Animal and tumor models
Six to eight-week-old female wild-type C57BL/6 mice (strain #000664) were from
Jackson
Laboratory (Sacramento, CA, USA). Mice were shaved at sites of subcutaneous
tumor
transplantation prior to injection. Tumors were established via subcutaneous
injection of 6 x 106
cells for each model. In some experiments, multiple MC38 tumors were
established. Established
tumors were treated at volumes of approximately 50 min' (4 to 5 mm in
diameter; day 4 for MC38-
luc and LLC tumors; day 18 for MOC1 tumors). For NIR-PIT treatments and
fluorescence/bioluminescence imaging (BLI), mice were anesthetized with
inhaled 3-5% isoflurane
and/or via intraperitoneal injection of 1 mg of sodium pentobarbital (Nembutal
Sodium Solution,
Ovation Pharmaceuticals Inc., Deerfield, IL, USA). CD44-IR700 was administered
via IV (tail-
vein) injection and NIR light was administered at 50 J/cm2 on day 5 and 100
J/cm2 on day 6.
Previous results demonstrated that two NIR light doses kill up to 80% of
target-expressing cells
(Mitsunaga et al., Nat Med. 2011; 17:1685-91; Nagaya et al., Mol Cancer Res.
2017; 15:1667-77).
For mice bearing multiple tumors, tumors not exposed to NIR were shielded from
NIR light
exposure with aluminum foil. PD-1 mAb was administered via intraperitoneal
injection using
standard technique. Tumor volumes were based on caliper measurements (tumor
volume = length
x width2 x 0.5). In some MC38 experiments, mice cured after combination NIR-
PIT and PD-1
mAb treatment were challenged via subcutaneous injection of MC38 (6 x 106)
cells in the
contralateral flank. Tumor volume and animal weight was measured three times a
week for MC38-
luc and LLC tumors and two times a week for MOC1 tumor until the tumor volume
reached 2000
min3, whereupon the mice were euthanized with inhalation of carbon dioxide
gas. For all immune
correlative experiments, mice were euthanized via awake cervical dislocation.
Fluorescence imaging
In vitro, MC38-luc, LLC or MOC1 cells (1 x 104) were seeded on cover-glass-
bottom
dishes, incubated for 24 h, then exposed to 10 lig/mL CD44-IR700 for 6 h at 37
C. Cells were then
analyzed via fluorescence microscopy (BX61; Olympus America, Inc., Melville,
NY, USA) using a
590-650 nm excitation filter and a 665-740 nm band pass emission filter.
Transmitted light
differential interference contrast (DIC) images were also acquired. In vivo,
IR700 fluorescence and
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white light images were obtained using a Pearl Imager (700 nm fluorescence
channel) and analyzed
using Pearl Cam Software (LICOR Biosciences, Lincoln, NE). Regions of interest
(ROIs) within
the tumor were compared to adjacent non-tumor regions as background (left
dorsum). Average
fluorescence intensity of each ROI was calculated. (n 10).
Bioluminescence imaging (BLI)
In vitro, MC38-luc cells were seeded into 12 well plates (2 x 105 cells/well)
or a 10 cm dish
(2 x 107 cells), incubated for 24 h, then exposed to 10 pg/mL of CD44-IR700
for 6 h at 37 C. Cells
were treated with LED or NIR laser light (690 5 nm, BWF5-690-8-600-0.37; B&W
TEK INC.,
Newark, DE, USA) in phenol-red-free culture medium. For luciferase activity,
cells were exposed
to 150 pg/mL D-luciferin (Gold Biotechnology, St. Louis, MO, USA) 1 h after
NIR-PIT treatment,
and luciferase activity (photons/min) was obtained on a BLI system (Photon
Imager; Biospace Lab,
Paris, France) using M3 Vision Software (Biospace Lab). In vivo, D-luciferin
(15 mg/mL, 200 pL)
was injected intraperitoneally and the mice were analyzed on a BLI system
(Photon Imager) for
luciferase activity (photons/min/cm2). ROIs were set to include the entire
tumor with the adjacent
non-tumor region as background.
Histological analysis
Tumors (day 10 for MC38-luc and LLC tumors, day 24 for MOC1 tumors) were
excised,
formalin-fixed and paraffin embedded, and sectioned at 10 1.tm. Following
standard H&E staining,
sectioned were analyzed on an Olympus BX61 microscope.
Immunofluorescence
Formalin fixed paraffin embedded sections were stained as described (18).
Briefly, sections
were deparaffinized in an ethanol gradient, then blocked in separate
incubations with bloxall
(Vector Laboratories), 2.5% normal goat serum (Vector Laboratories) and
Renaissance Ab diluent
(Biocare Medical). Primary antibody targeting CD4 (Invitrogen, clone 45M95,
1:75 dilution) in
Renaissance Ab diluent was added for 45 minutes on an orbital shaker. Slides
were washed five
times then stained with an anti-rat secondary antibody (Vector Laboratories).
Following four more
washes, slides were stained with TSA conjugated 0pa1650 (Perkin Elmer, 1:150
dilution) in
Amplification plus buffer (Perkin Elmer). Slides were washed four times with
1X TBS-T. Slides
were washed, exposed to antigen stripping buffer (0.1 M glycine pH10 + 0.5%
tween 20), and re-
blocked as above. Primary antibody targeting CD8 (Invitrogen, clone 415, 1:75
dilution) in
Reinassance Ab diluent was added for 45 minutes. A nti-rat secondary antibody
(Vector
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Laboratories) was added as above. Following four more washes, slides were
stained with TSA
conjugated 0pa1520 (Perkin Elmer, 1:150 dilution) in Amplification plus buffer
(Perkin Elmer).
Nuclei counter-staining was achieved with Spectral DAPI (Perkin Elmer, 1:500).
Slides were
rinsed once with ddH20, coverslipped with Vectashield hard mount (Vector
Laboratories) and
sealed with nail polish.
Flow cytometry
In vitro, MC38-luc, LLC or MOC1 cells (2 x 105) were seeded into 12 well
plates and
incubated for 24 h then exposed to media containing 10 ug/mL of CD44-IR700 for
6 h at 37 C.
Cells were harvested and analyzed on a BD FACSCalibur (BD Biosciences) using
CellQuest
software. To validate specific binding of CD44-IR700, cells were incubated
with excess
unconjugated CD44 antibody (100 ug) prior to incubation with CD44-IR700. In
vivo, tumors were
harvested (day 10 for MC38-luc and LLC tumors, day 24 for MOC1 tumors) and
immediately
digested as previously described (Moore et al., Cancer Immunol Res. 2016;
4:1061-71). Following
FcyR (CD16/32) block, single cell suspensions were stained with primary
antibodies. Suspensions
were stained with fluorophore-conjugated primary antibodies including anti-
mouse CD45.2 clone
104, CD3 clone 145-2C11, CD8 clone 53-6.7, CD4 clone GK1.5, PD-1 clone
29F.1Al2, CD11c
clone N418, F4/80 clone BM8, CD1lb clone M1/70, Ly-6C clone HK1.4, Ly-6G clone
1A8, I-A/I-
E clone M5/114.15.2, PD-Li clone 10F.9G2, CD25 clone PC61.5.3, CTLA-4 clone
UC10-4B9,
CD31 clone 390, PDGFR clone APA5 and CD44 clone IM7 (Biolegend) for one hour
in a 1%
BSA/1xPBS buffer. Suspensions were washed, stained with a viability marker
(7AAD or zombie
dye; Biolegend) and analyzed by flow cytometry on a BD Canto using BD FACS
Diva software.
Isotype controls and "fluorescence minus one" methods were used to validate
staining specificity.
FoxP3+ regulatory CD4+ T-lymphocytes (Legs) were stained using the Mouse
Regulatory T Cell
Staining Kit #1 (eBioscience) per manufacturer protocol. Post-acquisition
analysis was performed
with FlowJo vX10Ø7r2.
Antigen-specific TIL reactivity
Minced fragments of fresh tumor were incubated in RPMI 1640-based media
supplemented
with glutamine, HEPES, nonessential amino acids, sodium pyruvate, 13-
mercaptoethanol, 5% 1-BS,
and 100 U/mL recombinant murine IL-2 for 72 hours to extract TIL. Untouched
TIL were enriched
with negative magnetic sorting (AutoMACSpro, Miltenyi Biotec). Antigen
presenting cells (APC;
splenocytes from naive, WT B5 mice irradiated to 50Gy) were pulsed for one
hour with peptides of
interest including class I-restricted antigens p1 5E604611 (H-2Kb-restricted
KSPWFTTL),
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Survivin/Birc 5 57_64 (H-2Kb-restricted QCFFCFKEL), Twist 125_133 (H-2Db-
restricted TQSLNEAFA),
and Trp53232-24o (H-2Db-restricted KYMCNSSCM) Antigen-pulsed APC and TIL were
co-
incubated for 24 hours at a 3:1 APC:TIL ratio. Supernatants were analyzed for
IFNy production by
ELISA (R&D) per manufacturer recommendations. TIL alone, APC alone, and
peptide
.. stimulations with 0Va1bUlnin257-264 (H-2Kb-restricted SIINFEKL) and VSV-N52-
59 (H-2Db-restricted
RGYVYQGL) were used as controls.
RT-PCR
RNA from whole tumor lysates was purified using the RNEasy Mini Kit (Qiagen)
per the
manufacturer's protocol. cDNA was synthesized utilizing a high capacity cDNA
reverse
transcription kit with RNase inhibitor (Applied Biosystems). A Taqman
Universal PCR master
mix was used to assess the relative expression of target genes compared to
GAPDH on a Viia7
qPCR analyzer (Applied Biosystems). Custom primers were designed to flank
nucleotide regions
encoding the MHC class I-restricted epitopes for each tumor associated
antigen.
Statistical analysis
Data are expressed as means SEM from a minimum of five experiments, unless
otherwise
indicated. Statistical analyses were carried out using GraphPad Prism version
7 (GraphPad
Software, La Jolla, CA, USA). Student's t test was used to compare the
treatment effects with that
of control in vitro. To compare tumor growth in a re-inoculated tumor model of
MC38-luc, the
Mann Whitney test was used. For multiple comparisons, a one-way analysis of
variance (ANOVA)
followed by the Tukey's test was used. The cumulative probability of survival
based on volume
(2000 mm3) were estimated in each group with a Kaplan-Meier survival curve
analysis, and the
results were compared with use of the log-rank test. A p-value of < 0.05 was
considered statistically
significant.
Example 2
In vitro Effects of NIR-PIT on Cancer Cells
MC38-luc is a mouse colon cancer cell line expressing luciferase under control
of the CMV
promoter (Zabala et al., Mol. Cancer 8:2, 2009). LLC (Lewis lung carcinoma)
cells and MOC1
(murine oral carcinoma) cells were also used. Anti-CD44-IR700 was produced
using the methods
described in WO 2013/009475 (incorporated by reference herein). Briefly, anti-
CD44 mAb (1.0
mg, 6.7 nmol, clone IM7 from BioXCell, West Lebanon, NH) was incubated with
IR700 NHS ester
(65.1 ug, 33.3 nmol) in 0.1 M Na2HPO4 (pH 8.6) at room temperature for 1 h and
purified with a
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Sephadex G25 column (PD-10; GE Healthcare, Piscataway, NJ, USA). CD44-IR700
conjugate
synthesis was controlled so that an average of two IR700 molecules were bound
to each CD44
antibody. Conjugates demonstrated strong fluorescent intensity and peak
absorbance around 690
nm.
The effect of anti-CD44-IR700 on MC38-luc cells was evaluated in vitro. To
verify anti-
CD44¨IR700 binding, fluorescence from cells after incubation with anti-
CD44¨IR700 was
measured using a flow cytometer (FACS Calibur, BD BioSciences) and CellQuest
software (BD
BioSciences). MC38-luc cells were seeded into 12-well plates and incubated for
24 hours.
Medium was replaced with fresh culture medium containing 10 mg/mL of anti-
CD44¨IR700 and
incubated for 6 hours at 37 C. To validate the specific binding of the
conjugated antibody, excess
antibody (100 mg) was used to block 10 mg of anti-CD44¨IR700 (FIG. 1A).
To detect the antigen specific localization and effect of NIR-PIT,
fluorescence microscopy
was performed (BX61; Olympus America, Inc.). MC38-luc, LLC or MOC1 cells (1 x
104) were
seeded on cover-glass-bottomed dishes and incubated for 24 hours. Anti-
CD44¨IR700 was then
added to the culture medium at 10 mg/mL and incubated for 6 hours at 37 C.
After incubation, the
cells were washed with phosphate buffered saline (PBS). The filter set to
detect IR700 consisted of
a 590 to 650 nm excitation filter, a 665 to 740 nm band pass emission filter.
Transmitted light
differential interference contrast (DIC) images were also acquired. FIG. 1B is
a digital image
showing differential interference contrast (DIC) and fluorescence microscopy
images of control and
anti-CD44-IR700 treated MC38-luc cells. Necrotic cell death was observed upon
excitation with
NIR light in treated cells. This signal was completely reversed in the
presence of excess
unconjugated CD44 mAb, verifying binding specificity. NIR light exposure of
tumor cells exposed
to CD44-IR700 induced immediate cellular swelling, bleb formation, and rupture
of vesicles
indicative of necrotic cell death in all three cell lines (MC38-luc, LLC, and
MOC1). These
morphologic changes were observed within 15 mm of NIR exposure (FIG. 1B).
For bioluminescence imaging (BLI), MC38-luc cells were seeded into 12 well
plates (2 x
105 cells/well) or a 10 cm dish (2 x 107 cells)were seeded onto a 10-cm dish
and preincubated for
24 hours. After replacing the medium with fresh culture medium containing 10
mg/mL of anti-
CD44¨IR700, the cells were incubated for 6 hours at 37 C in a humidified
incubator. After
washing with PBS, phenol-red-free culture medium was added. Then, cells were
exposed with a
LED or a NIR laser which emits light at a 685 to 695 nm wavelength (BWF5-690-8-
600-0.37;
B&W TEK INC.) in phenol-red-free culture medium. The output power density in
mW/cm2 was
measured with an optical power meter (PM 100, Thorlabs). FIG. 1C is a digital
image of
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bioluminescence imaging (BLI) of a 10-cm dish showing NIR-light dose dependent
luciferase
activity in MC38-luc cells.
For luciferase activity (FIG. 1D), 150 mg/mL D-luciferin-containing media
(Gold
Biotechnology) was administered to PBS-washed cells 1 hour after NIR-PIT and
images were
obtained on a BLI system (Photon Imager; Biospace Lab). Regions of interest
(ROI) were placed
on each entire well, and the luciferase activity (photons/min) was then
calculated using M3 Vision
Software (Biospace Lab).
The cytotoxic effects of NIR-PIT with anti-CD44¨IR700 were determined by flow
cytometric propidium iodide (PI; Life Technologies) staining, which can detect
compromised cell
membranes. Two hundred thousand MC38-luc cells were seeded into 12-well plates
and incubated
for 24 hours. Medium was replaced with fresh culture medium containing 10
mg/mL of anti-
CD44¨IR700 and incubated for 6 h at 37 C. After washing with PBS, PBS was
added, and cells
were irradiated with a red light-emitting diode (LED), which emits light at
670 to 710 nm
wavelength (L690-66-60; Marubeni America Co.) at a power density of 50 mW/cm2
as measured
with an optical power meter (PM 100, Thorlabs). Cells were scratched 1 hour
after treatment. PI
was then added in the cell suspension (final 2 mg/mL) and incubated at room
temperature for 30
minutes, followed by flow cytometry. Each value represents mean SEM of five
experiments.
FIG. 1E shows percentage of cell death in MC38-luc cells treated with NIR with
or without 10
pg/ml CD44-IR700, measured with dead cell count using propidium iodide (PI)
staining.
Bioluminescence imaging demonstrated decreased luciferase activity in a light-
dose
dependent manner (FIGS. 1C, 1D) in MC38-luc cells. Based on incorporation of
propidium iodine
(e.g., membrane permeability), NIR induced cell death in a light-dose
dependent manner in MC38-
luc (FIG. 1E), LLC (FIG. 1F) and MOC1 (FIG. 1G) cells exposed to CD44-IR700.
NIR or CD44-
1R700 alone did not induce significant alterations in cell viability.
These data demonstrate that NIR-PIT targeting CD44 induced specific cell death
in MC38-
luc, LLC and MOC1 cells in vitro.
Example 3
CD44 expression within MOC1, LLC and MC38-luc Tumor Compartments
To verify target expression of CD44 in vivo, size matched MOC1 (day 24), LLC
(day 10)
and MC38 (day 10) tumors were assessed for CD44 expression within different
tumor
compartments via flow cytometry (FIG. 2A). Significant heterogeneity in tumor
and stromal cell-
specific CD44 expression was observed, with LLC and MC38-luc tumor cells
expressing
significantly greater levels of CD44 compared to MOC1. Expression of CD44 on
immune cell
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subsets was more homogeneous between MOC1, LLC and MC38-luc tumors and was
greater than
CD44 expression on tumor and stromal cells on a cell-by-cell basis as measured
by mean
fluorescence intensity (MFI). Whole tumor accumulation of CD44-IR700 one day
after injection,
which is dependent upon multiple factors including target antigen expression
and vascularity, was
significantly greater in MC38-luc tumors (p<0.001) compared to LLC or MOC1
tumors (FIGS. 2B,
2C).
Example 4
In vivo Effects of NIR-PIT and PD-1 mAb on Tumors
The effect of a combination therapy with anti-CD44-IR700 and anti-PD1 was
tested in
unilateral, bilateral, and multiple tumor models in mice. FIG. 3A shows the
treatment scheme for a
unilateral MC38-luc tumor in mice (10-13 mice in each group). Mice were
unilaterally injected in
the flank with 6 million tumor cells (day 0). Established tumors were treated
at volumes of
approximately 50 min3 (4 to 5 mm in diameter; day 4 for MC38-luc and LLC
tumors; day 18 for
MOC1 tumors). On day 4, the mice were administered 100 pg anti-CD44-IR700 i.v.
(tail vein)
alone, or in combination with 200 pg anti-PD1 i.p. (within 1 hour of one
another) (anti-mouse PD-1
(CD279) specific mAb (clone RMP1-14) from BioXCell (West Lebanon, NH, USA))
and were
subsequently administered 100 pg anti-PD1 i.p. on days 6, 8, and 10. NIR-PIT
was performed on
days 5 (50 J/cm2) and 6 (100 J/cm2). For mice bearing multiple tumors, tumors
not exposed to NIR
were shielded from NIR light exposure with aluminum foil.
Tumors were monitored by fluorescence imaging and bioluminescence imaging
(FIG. 3A).
in vivo IR700 fluorescence images were obtained with a Pearl Imager (LI-COR
Biosciences) with a
700-nm fluorescence channel. A ROI was placed on the tumor and the average
fluorescence
intensity of IR700 signal was calculated for each ROI using a Pearl Cam
Software (LICOR
Biosciences). For in vivo BLI, D-luciferin (15 mg/mL, 200 L) was injected
i.p., and the mice
were analyzed on a BLI system (Photon Imager) for luciferase activity. ROIs
were set to include
the entire tumor in order to quantify BLI. ROIs were also placed in the
adjacent non-tumor region
as background (photons/min/cm2). Average luciferase activity of each ROI was
calculated.
To detect the antigen-specific microdistribution in the tumor, fluorescence
microscopy was
performed. Tumor xenografts were excised (day 10 for MC38-luc and LLC tumors,
day 24 for
MOC1 tumors) from the right flank xenograft without treatment. Extracted
tumors were frozen
with optimal cutting temperature (OCT) compound (SAKURA Finetek Japan Co.) and
frozen
sections (10 pin thick) prepared. Fluorescence microscopy was performed using
an Olympus
BX61 microscope with the following filters: excitation wavelength 590 to 650
nm, emission
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wavelength 665 to 740 nm long pass for IR700 fluorescence. DIC images were
also acquired. To
evaluate histological changes, light microscopy was performed using Olympus
BX61. Tumor
xenografts were excised from mice without treatment, 24 hours after injection
of anti-CD44¨IR700
(i.v.) and 24 hours after NIR-PIT. Tumors were also excised from mice with
bilateral flank tumors
.. (both treated right-sided tumors and untreated left-sided tumors) 24 hours
after NIR-PIT of the
right tumor. Extracted tumors were also placed in 10% formalin, and serial 10-
mm slice sections
were fixed on a glass slide with H&E staining.
Compared to control or PD-1 mAb alone groups, NIR-PIT resulted in a near-
immediate
decrease in tumor fluorescence signal, likely due to dispersion of IR700 from
dying cells (FIG. 3B).
.. Combination NIR-PIT and PD-1 mAb treatment resulted in dramatically
decreased
bioluminescence compared to control or single treatment groups (FIG. 3C,
quantified in FIG. 3D).
Histologic (H&E) analysis of treated tumors revealed extensive tumor necrosis
and micro-
hemorrhage in tumors treated with NIR-PIT, while groups treated with PD-1 mAb
demonstrated
greater leukocyte infiltration (FIG. 3E). While primary tumor growth was
inhibited following NIR-
PIT or PD-1 mAb alone compared to control (FIG. 3F), combination treatment
resulted in
significant tumor control and complete rejection of established MC38-luc
tumors in 9 of 13 (70%)
mice. This response resulted in significantly prolonged survival in mice
receiving combination
treatment (FIG. 3G). While antibody treatment or anti-CD44-IR700 NIR-PIT
increased survival
time compared to control, none of the animals in the NIR-PIT group survived to
40 days and only
9% of those in the antibody only group (anti-CD44-IR700 + anti-PD1 without NIR-
PIT) survived
to the end of the study (FIG. 3G). In contrast, 80% of the animals in the
combination treatment
group survived to the end of the study. Neither skin necrosis nor systemic
toxicity was observed
within any treatment group.
Similar approaches were taken in mice bearing established unilateral LLC or
MOC1 tumors
.. using similar treatment regimens and imaging protocols (FIGS. 4A, 5A).
Similar to MC38-luc
tumors, treatment of LLC or MOC1 tumors with NIR-PIT resulted in near-
immediate loss of IR700
fluorescent signal (FIGS. 4B, 5B) indicating on-target effects. Treatment of
LLC tumor-bearing
mice with combination NIR-PIT and PD-1 mAb significantly enhanced primary
tumor control
(FIG. 4C) and survival (FIG. 4D) over control or either treatment alone, and
resulted in rejection of
1 of 12 (8%) established tumors. Treatment of MOC1 tumor-bearing mice with
combination NIR-
PIT and PD-1 mAb induced rejection of 1 of 13 (8%) established tumors and
resulted in
statistically enhanced survival compared to control, but cumulative primary
tumor growth
following combination treatment was not enhanced over either treatment alone
(FIGS. 5C, 5D).
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Taken together, these results demonstrate CD44 on-target effects of NIR-PIT in
MC38-luc,
LLC and MOC1 tumor-bearing mice, with significant enhancement of primary tumor
control and
survival with the addition of PD-1 immune checkpoint blockade (ICB) in the
MC38-luc and LLC
models.
Example 5
Enhancement of antigen-specific immunity induction with
NIR-PIT by PD-1 ICB
Following completion of treatment, some MC38-luc tumors were processed into
single cell
suspensions and assessed for infiltration of immune cells with flow cytometry.
Tumors treated
with NIR-PIT demonstrated significantly enhanced infiltration by CD8 and CD4
tumor infiltrating
lymphocytes (TIL) (FIG. 6A) that expressed greater levels of PD-1. Mice
treated with systemic
PD-1 mAb demonstrated PD-1 target saturation as very low levels of PD-1 were
detectible on the
surface of TIL from these tumors by flow cytometry after staining with the
same Ab clone (RMP1-
14). This enhanced CD8 and CD4 TIL infiltration was verified by multiplex
immunofluorescence
(IF). In control or PD-1 mAb treated tumors, few CD8+ TIL nested along the
tumor-stromal
interface but did not infiltrate the tumor (FIG. 6B, left panels). Following
NIR-PIT, more CD8+
TIL infiltrated throughout the tumor but many TIL were still arrested at the
tumor-stromal
interface. Infiltration into the tumor was significantly enhanced with the
addition of PD-1 mAb
(FIG. 6B, right panels). In additional experiments, TIL were extracted from
control or treated
MC38-luc tumors via IL-2, and assessed for antigen-specific IFNy responses to
multiple H-2Kb or
H-2Kd-restricted TAA (FIG. 6C). TIL from control tumors demonstrated
measurable responses to
H-2Kb-restricted pl5E604_611 (KSPWFTTL) but lacked responses to other
antigens. PD-1 mAb
treatment enhanced the baseline pl5E604_611 responses but did not induce
responses against other
antigens. NIR-PIT treatment induced de novo responses that were absent at
baseline to H-2Kb-
restricted Survivin/Birc557_64 (QCFFCFKEL) and H-2Db-restricted Trp5 3232_240
(KYMCNSSCM)
and enhanced baseline responses to pl5E604_611. Treatment with PD-1 mAb
enhanced these NIR-
PIT induced or enhanced antigen-specific responses. NIR-PIT also enhanced
tumor infiltration of
MHC class II-positive dendritic cells (DCs) and F4/80+ macrophages polarized
to express greater
levels of MHC class II (FIG. 6D). Immunosuppressive neutrophilic-myeloid (PMN-
myeloid) and
regulatory CD4+ T-lymphocytes (Tõgs) were variably altered by combination
treatment (FIG. 6E).
MC38-luc tumor cell specific PD-Li expression was verified but did not change
with treatment,
while infiltrating immune cell PD-Li was significantly greater than tumor cell
expression, and
increased with combination treatment (FIG. 6F).
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Similar immune correlative experiments were carried out in LLC and MOC1
tumors. LLC
tumors treated with PD-1 mAb and NIR-PIT alone or in combination demonstrated
enhanced TIL
infiltration (FIG. 7A). Antigen-specific LLC TIL demonstrated measureable
baseline responses to
pl5E6o4-611 and H-2Db-restricted Twist125-133 (TQSLNEAFA). Similar to MC38-luc
tumors, NIR-
PIT treatment induced responses to Survivin/Birc557_64. Responses to Birc5 and
Twist but not pl5E
were enhanced with PD-1 mAb treatment (FIG. 7B). NIR-PIT treatment of LLC
tumors enhanced
infiltration of MHC class II-positive DCs and MHC class II expression on
macrophages (FIG. 7C).
PMN-myeloid cells and Legs were variably altered following treatments FIG.
7D), and LLC tumor
and immune cell-specific PD-Li expression was enhanced with treatment (FIG.
7E).
In contrast to MC38-luc or LLC tumors, MOC1 tumors treated with NIR-PIT
demonstrated
few immune correlative alterations. CD8 and CD4 TIL infiltration was modestly
enhanced with
PD-1 mAb but not NIR-PIT (FIG. 8A). Baseline TIL antigen-specific responses to
pl5E6o4-611
were enhanced with systemic PD-1 mAb treatment, but responses to other shared
tumor antigens
were not induced with NIR-PIT treatment (FIG. 8B). MOC1 tumor infiltration of
MHC class II+
DCs and macrophages was modestly enhanced, indicating a lack of myeloid cell
priming and
activation in this model. No significant changes were observed in infiltration
of PMN-myeloid
cells or Tregs or MOC1 tumor or immune cell-specific PD-Li expression (FIGS.
8C, 8D).
To investigate possible explanations for the lack of TIL responses against
tumor associated
antigens in MOC1, relative expression of each antigen was measured within MC38-
luc, LLC and
MOC1 cells. Using primers designed to flank the MHC class I-restricted epitope
coding region,
PCR results indicated low expression of Birc5, Twist] and Trp53 gene
transcripts in MOC1 relative
to MC38-luc and LLC (FIG. 9). Greater antigen expression generally correlated
with baseline TIL
responses. Interestingly, higher relative Trp53 expression in MC38-luc cells
and Twist] expression
in LLC cells correlated to enhanced TIL responses against the class I-
restricted epitopes from these
genes after combination NIR-PIT and PD-1 mAb treatment. Thus enhanced TIL
responses after
treatment may be dependent on baseline tumor antigen expression.
These results indicate that NIR-PIT can induce de novo, polyclonal antigen-
specific TIL
responses against MHC class I-restricted tumor antigens in MC38-luc and LLC
tumor bearing
mice, and that these responses can be enhanced with systemic PD-1 ICB.
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Example 6
Combination NIR-PIT and PD-1 ICB induced
Abscopal anti-tumor effect in mice bearing bilateral MC38-luc tumors
Given evidence of induction of tumor antigen-specific immunity following NIR-
PIT in
MC38-luc tumor bearing mice, whether local NIR-PIT combined with systemic PD-1
mAb could
induce anti-tumor immunity in a separate, distant tumor not treated with NIR-
PIT was determined.
Treatment and imaging regimens (FIG. 10A) were similar for mice bearing
bilateral MC38-luc
tumors as described above, but only the right flank tumor was treated with NIR-
PIT (FIG. 10B).
NIR-PIT induced near-immediate loss of IR700 fluorescent signal in the treated
tumor,
whereas loss of IR700 signal intensity in the untreated tumor was delayed for
several days (FIG.
10C). Conversely, bioluminescence of both right (treated with NIR-PIT) and
left (untreated)
MC38-luc tumors decreased concurrently after combination treatment (FIG. 10D,
quantified in
FIG. 10E). Histologic analysis of both right and left tumors revealed similar
patterns of necrosis
and micro-hemorrhage and increase leukocyte infiltration (FIG. 10F).
Combination treatment
resulted in significant primary tumor control and complete tumor rejection of
both right and left
tumors in 8 of 10 mice (80%; FIG. 10G), leading to enhanced survival compared
to untreated mice
(FIG. 10H).
Example 7
Induction of antigen-specific immunity in distant tumors not treated with NIR-
PIT
Flow cytometric analysis of single cell suspensions from both right (treated
with NIR-PIT)
and left (untreated) tumors revealed similar levels of enhanced CD8 and CD4
TIL accumulation
(FIG. 11A). Assessment of antigen-specific reactivity demonstrated that TIL
from both treated and
untreated tumors reacted to the same MHC class I-restricted antigens (FIG.
11B), indicating the
presence of systemic antigen-specific immunity. TIL responses were similar in
magnitude to
pl5E6o4-611 and Survivin/Birc557-64, but responses to Trp53232-240 were
diminished in tumors not
treated with NIR-PIT compared to those treated. Increased MHC class II-
positive DC and
macrophages (FIG. 11C), increased PMN-myeloid cells and decreased 'Legs (FIG.
11D) were
observed in treated but not untreated tumors, indicating these changes are a
direct result of NIR-PIT
and not a result of systemic anti-tumor immunity. MC38-luc infiltrating immune
cell PD-Li
expression FIG. 11E) was enhanced in both right treated and left untreated
tumors in mice
receiving combination treatment, indicating that immune cell PD-Li expression
may be
independent of NIR-PIT.
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Thus, combination NIR-PIT and PD-1 ICB can lead to the development of systemic
tumor
antigen-specific immunity capable of eliminating an established untreated
tumor, but enhanced
innate immunity and alterations in immunosuppressive cell subsets appear to
occur locally as a
more direct effect of NIR-PIT.
Example 8
Combination NIR-PIT and PD-1 ICB Controls
multiple distant tumors in mice with high disease burden
To demonstrate that treatment of a single MC38-luc tumor could lead to
rejection of
multiple established distant tumors within an individual mouse, the following
methods were used.
Similar treatments (FIG. 12A) were used to deliver NIR-PIT to one of four
established MC38
tumors (FIG. 12B). NIR-PIT induced near-immediate loss of IR700 fluorescent
signal in the single
treated tumor, whereas resolution of IR700 signal intensity in the three
untreated tumors was
delayed for several days (FIG. 12C). Conversely, bioluminescence of both the
single treated and
three untreated MC38-luc tumors decreased concurrently after combination
treatment (FIG. 12D,
quantified in FIG. 12E). Histologic analysis revealed necrosis and increased
leukocyte infiltration
in all tumors from treated mice but not tumors from control mice (FIG. 12F).
Systemic PD-1 mAb
and treatment of a single MC38-luc tumor with NIR-PIT resulted in dramatic
growth control
multiple MC38-luc tumors. Twelve of 15 (80%) treated mice (FIG. 12G)
completely rejected all
four tumors, resulting in enhanced survival compared to control (FIG. 12H).
Thus, treatment of a single focus of tumor with local NIR-PIT plus systemic PD-
1 ICB is
sufficient to induce systemic immunity capable of eliminating multiple sites
of distant disease not
treated with NIR-PIT.
Example 9
Mice that rejected tumors after combination NIR-PIT and PD-1 ICB
developed immunologic memory
To assess for the presence of immunologic memory, mice were treated with NIR-
PIT and
PD-1 mAb as described above (FIG. 13A). Mice that demonstrated a complete
response to
combination treatment were challenged 30 days later with injection of MC38-luc
cells in the
contralateral flank (FIG. 13A). Whereas control mice readily were engrafted
with MC38-luc
tumors, mice that previously rejected established MC38-luc tumors resisted
engraftment and did
not grow tumors (FIG. 13C, survival in FIG. 13D), demonstrating the presence
of immunologic
memory.
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As depicted in FIG. 18, the results in the examples above demonstrate that NIR-
PIT
induces CD44-specific tumor cell death, leading to the release of multiple
tumor antigens. NIR-
PIT also promotes a pro-inflammatory tumor microenvironment, resulting in
cross priming of
multiple antigens and the development of a polyclonal antigen-specific T-cell
response. This
effector response is limited by PD-1/PD-L1 expression and adaptive immune
resistance, which is
effectively reversed with the addition of PD-1 ICB.
Example 10
Materials and Methods
This example provides the materials and methods used to obtain the results
described in
Examples 11-14.
Cell culture
MC38-luc cells expressing CD44 and luciferase, LL/2 cells and MOC1 cells
stably
expressing CD44 antigen were cultured in RPMI1640 supplemented with 10% fetal
bovine serum
and 1% penicillin-streptomycin in tissue culture flasks in a humidified
incubator at 37 C in an
atmosphere of 95% air and 5% carbon dioxide.
Reagents
Water soluble, silica-phthalocyanine derivative, IRDye700DX NHS ester was from
LI-COR
Bioscience (Lincoln, NE, USA). An anti-mouse/human CD44 mAb (IM7) and anti-
mouse CD25
mAb (PC-61.5.3) were from Bio X Cell. All other chemicals were of reagent
grade.
Synthesis of IR700-conjugated anti-CD25 mAb and anti-CD44 mAb
Anti-CD25 mAb (lmg, 6.7nm01/L) and Anti-CD44 mAb (lmg, 6.7 nmol/L) were
respectively incubated with IR700 (65.1 pg, 33.3 nmol, 10 mmol/L in DMSO) and
0.1 mol/L
Na2HPO4 (pH 8.5) at room temperature for 1 hour. The mixture was purified with
a gel filtration
column (Sephadex G 25 column, PD-10, GE Healthcare, Piscataway, NJ, USA). The
protein
concentration was determined with Coomassie Plus protein assay kit (Thermo
Fisher Scientific Inc,
Rockford, IL, USA) by measurement of the absorption at 595 nm with
spectroscopy (8453 Value
System; Agilent Technologies, Santa Clara, CA, USA). Herein, IR700-conjugated
anti-CD25 mAb
and anti-CD44 mAb are abbreviated as anti-CD25-mAb-IR700 and anti-CD44-mAb-
IR700,
respectively.
Animal model
Six- to eight-week-old female C57BL/6 mice (strain #000664) were purchased
from the
Jackson laboratory. The lower part of the body of the mice was shaved for
irradiation and image
analysis. Mice with tumors reaching approximately 150 mm in volume were used
for the
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experiments. Tumor volumes were calculated from the greatest longitudinal
diameter (length) and
the greatest transverse diameter (width) using the following formula; tumor
volume = length x
width2 x 0.5, based on caliper measurements. Mice were monitored each day and
tumor volumes
were measured three times a week for MC38-luc and LL/2 tumors and twice a week
for MOC1
tumors until the tumor volume reached 2,000 mm3, whereupon the mice were
euthanized with
inhalation of carbon dioxide gas.
In vivo bioluminescence imaging (BLI) and IR700 fluorescence imaging
To obtain bioluminescence images in MC38-luc tumor-bearing mice, D-luciferin
(15
mg/mL, 150 pL) was intraperitoneally injected to mice. Luciferase activity was
analyzed with a
BLI system (Photon Imager; Biospace Lab, Paris, France) using relative light
units (RLU). Regions
of interest (ROI) were placed over the entire tumor. The counts per minute of
RLU were calculated
using M3 Vision Software (Biospace Lab) and converted to the percentage based
on RLU before
NIR-PIT (%RLU). BLI was performed before and after NIR-PIT on day 0 to day 7.
In vivo IR700
fluorescence images were obtained with a Pearl Imager (LI-COR Biosciences)
with a 700-nm
fluorescence channel.
In vivo fluorescence imaging studies
MC38-luc cells (8 million), LL/2 cells (8 million) and MOC1 cells (4 million)
were
subcutaneously injected in the dorsum of the mice. Mice with tumors were
studied after they
reached volumes of approximately 150 mm3. Serial dorsal fluorescence images of
IR700 were
obtained with a Pearl Imager using a 700-nm fluorescence channel 1, 4, 6, 12,
24, and 48 hours
after intravenous injection of 100 pg of anti-CD25-mAb-IR700 via the tail
vein. Regions of
interest (ROI) were placed on the tumor and the adjacent non-tumor region as
background. The
mean value of fluorescence intensity (MFI) was calculated for each ROI. Target-
to-background
ratio (TBR) was calculated from fluorescence intensities of tumors and
fluorescence intensity of
background by the following formula; (fluorescence intensity of tumor) ¨
(fluorescence intensity of
background) / (fluorescence intensity of background).
NIR-PIT
MC38-luc cells (8 million), LL/2 cells (8 million) and MOC1 cells (4 million)
were
subcutaneously injected in the dorsum of mice. The mice with tumors which
reached volumes of
approximately 150 mm3 were selected and divided randomly into 4 experimental
groups for the
following treatments: (1) no treatment (control); (2) intravenous injection of
100 pg anti-CD25-
mAb-IR700 followed by external NIR light irradiation at 100 J/cmon day 0 (CD25-
targeted NIR-
PIT); (3) intravenous injection of 100 pg anti-CD44-mAb-IR700 followed by
external NIR light
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irradiation at 100 J/cm2 on day 0 (CD44-targeted NIR-PIT); and (4) intravenous
injection of 100 pg
anti-CD25-mAb-IR700 and 100 pg anti-CD44-mAb-IR700 (combined NIR-PIT).
For the mice with MC38-luc tumor, LL/2 tumor, and MOC1 tumor in the NIR-PIT
treated
groups, intravenous injection of the APCs was performed 5, 5, and 28 days
after tumor inoculation,
respectively, followed by external NIR light irradiation at 100 J/cm21 day
after APC injection. NIR
light was irradiated from above a targeted tumor in tumor-bearing mice using a
red light emitting
diode (LED), which emits light in the range of 670 to 710 nm wavelength (L690-
66-60; Marubeni
America Co.) at a power density of 50 mW/cm2 as measured with an optical power
meter (PM 100,
Thorlabs). IR700 absorbs light at approximately 690nm. IR700 fluorescence
images were obtained
.. before and after therapy.
Statistical Analysis
Quantitative data were expressed as means SEM. For multiple comparisons (?3
groups),
a one-way analysis of variance followed by the Tukey-Kramer test was used. The
cumulative
probability of survival was analyzed by the Kaplan-Meier survival curve
analysis, and the results
.. were compared with the Log-rank test. Statistical analysis was performed
with JMP 13 software
(SAS Institute, Cary, NC). A p value of less than 0.05 was considered
significant.
Example 11
In vivo fluorescence imaging after administration of anti-CD25-mAb-IR700
High fluorescence MFI was observed in MC38-luc, LL/2, and MOC1 1 hour after
anti-
CD25-mAb-IR700 (APC) injection, and fluorescence in all cell types gradually
increased until 24
hours post injection (FIGS. 14A and 14B). The fluorescence 48 hours after APC
injection
decreased compared to the fluorescence at 24 hours. The TBR of anti-CD25-mAb-
IR700 in all cell
types also gradually increased until 24 hours followed by a decrease in TBR 48
hours after
injection of the APC (FIG. 14C). The highest MFI and TBR were observed 24
hours after APC
injection; MC38-luc and LL/2 tumors showed higher value in MFI and TBR than
MOC1 tumors
(FIGS. 14B and 14C).
These data demonstrate the rationale for delivery of therapeutic NIR light
exposure 1 day
after APC injection for both CD25- and/or CD44-targeted NIR-PIT in the
examples below.
Example 12
Efficacy of combined CD25- and CD44-targeted NIR-PIT for MC38-luc tumor
FOXP3+CD25 CD4+ Treg cells are frequently found within tumors. In several
types of
cancers, decreased ratios of CD8+ T cells to FOXP3+CD25 CD4+ Treg cells in
tumor-infiltrating
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lymphocytes (TILs) can be associated with poor prognosis. CD25-targeted NIR-
PIT was used to
deplete tumor-infiltrating Treg cells within the tumor without eliminating
local effector cells or
Treg cells in other organs, resulting in reversal of the permissive tumor
microenvironment (TME)
by removing immunosuppressive cells in the TME and subsequent tumor killing to
enhance tumor
directed NIR-PIT (achieved with the CD44-targeted NIR-PIT).
The NIR-PIT regimen and imaging protocol are depicted in FIG. 15A. One day
after
injection of anti-CD25- and/or anti-CD44-mAb-IR700, the tumors were exposed to
100 J/cm2 of
NIR light via LED light. IR700 tumor fluorescence signal decreased due to
dispersion of
fluorophore from dying cells and partial photo-bleaching in all cases (FIG.
15B).
To investigate tumor-killing efficacy after NIR-PIT, bioluminescence imaging
(BLI) was
performed before and after NIR-PIT up to day 7 (FIG. 15C). BLI was
quantitatively evaluated as
the percentage of RLU based on pre-treatment RLU (RLU Post/RLU Pre x 100 =
%RLU). BLI is a
highly sensitive tool for evaluating tumor cells after NIR-PIT and its
intensity depends on the
catalysis of luciferin by luciferase mediated by oxygen, Mg' and ATP.
In most mice in the NIR-PIT-treated groups, % relative light units (%RLU)
greatly
decreased shortly after NIR-PIT and then gradually increased (FIG. 15C). This
pattern of %RLU
change is likely due to a large amount of initial cell killing followed by
slower regrowth of cells not
originally killed. In contrast, in some mice undergoing CD25-targeted NIR-PIT
and in the
combined NIR-PIT groups, luciferase activity greatly decreased shortly after
NIR-PIT and
thereafter disappeared (FIG. 15C). This pattern of %RLU change is likely due
to a large amount of
initial cell killing followed by complete remission of treated tumors due to
an enhanced immune
response.
Post-treatment %RLU in all the NIR-PIT treated groups was significantly lower
at all time
points after NIR-PIT than in the control group (p < 0.05, Tukey-Kramer test)
(FIG. 15D). In
addition, combined CD25- and CD44-targeted NIR-PIT showed significantly lower
%RLU 7 days
after NIR-PIT compared with CD44-targeted NIR-PIT alone (p <0.05, Tukey-Kramer
test) (FIG.
15D). These data indicate that combined CD25- and CD44-targeted NIR-PIT can
induce superior
in vivo tumor-killing effects compared to either APC alone. Tumor volume in
all the NIR-PIT
treated groups was significantly inhibited 5, 7 and 10 days after NIR-PIT
compared with that in the
.. control group (p <0.05, Tukey-Kramer test) (FIG. 15E) but the combined CD25-
and CD44-
targeted NIR-PIT showed significantly greater tumor reduction compared to CD44-
targeted NIR-
PIT alone at 7 and 10 days after NIR-PIT (p <0.05, Tukey-Kramer test) (FIG.
15E). No significant
tumor inhibition was observed in the other groups.
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These data indicate that combined CD25- and CD44-targeted NIR-PIT led to the
slowest
rate of tumor regrowth compared with other NIR light exposure groups. Combined
CD25- and
CD44-targeted NIR-PIT also was associated with significantly prolonged
survival after NIR-PIT
compared with CD25-targeted NIR-PIT alone (p < 0.05, Log-rank test) and CD44-
targeted NIR-
PIT alone (p < 0.01, Log-rank test) (FIG. 15F). Moreover, 8 of 14 mice in the
combined NIR-PIT
group achieved complete remission after a single round of NIR-PIT.
These results show that combined CD25- and CD44-targeted NIR-PIT enables
superior in
vivo therapeutic responses compared to the other two types of NIR-PIT for MC38-
luc tumors.
Example 13
Efficacy of combination with CD25- and CD44-targeted NIR-PIT for LL/2 tumor
The NIR-PIT regimen and imaging protocol are depicted in FIG. 16A. One day
after
injection of anti-CD25- and/or anti-CD44-mAb-IR700, the tumors were exposed to
100 J/cm2 of
NIR light. IR700 tumor fluorescence signal decreased due to dispersion of
fluorophore from dying
cells and partial photo-bleaching (FIG. 16B). Tumor volume in all the NIR-PIT
treated groups was
significantly inhibited 5, 7, 10 and 12 days after NIR-PIT compared to that in
the control group (p <
0.05, Tukey-Kramer test) (FIG. 16C). Among the three NIR-PIT treated groups,
combined CD25-
and CD44-targeted NIR-PIT showed significantly greater tumor reduction
compared to CD44-
targeted NIR-PIT alone 17 days after NIR-PIT (p < 0.05, Tukey-Kramer test)
(FIG. 16C). In the
long-term follow-up, combined CD25- and CD44-targeted NIR-PIT had
significantly prolonged
survival after NIR-PIT compared with CD25-targeted NIR-PIT alone or CD44-
targeted NIR-PIT
alone (p < 0.05, Log-rank test) (FIG. 16D). In 3 of 9 mice in the combined NIR-
PIT group
complete remission of tumor was achieved after only a single round of NIR-PIT.
Thus, combined CD25- and CD44-targeted NIR-PIT was therapeutically superior to
the
other 2 types of NIR-PIT in LL/2 tumors.
Example 14
Efficacy of combined CD25- and CD44-targeted NIR-PIT for MOC1 tumor
The NIR-PIT regimen and imaging protocol are depicted in FIG. 17A. One day
after
.. injection of anti-CD25- and/or anti-CD44-mAb-IR700, the tumors were exposed
to 100 J/cm2 of
NIR light. IR700 tumor fluorescence signal decreased due to dispersion of
fluorophore from dying
cells and partial photo-bleaching. (FIG. 17B). Tumor volume in all the NIR-PIT
treated groups
was significantly inhibited at all time points after NIR-PIT compared to the
control group (p < 0.05,
Tukey-Kramer test) (FIG. 17C). Combined CD25- and CD44-targeted NIR-PIT showed
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significantly greater tumor reduction 28 days after NIR-PIT compared to CD44-
targeted NIR-PIT
(p <0.05, Tukey-Kramer test).
In the long-term follow-up, combined CD25- and CD44-targeted NIR-PIT showed
significantly prolonged survival compared to CD44-targeted NIR-PIT (p <0.05,
Log-rank test)
(FIG. 17D). On the other hand, there was no significant difference in tumor
volume and survival
between CD25-targeted NIR-PIT alone and CD44-targeted NIR-PIT alone, and
between CD25-
targeted NIR-PIT alone and the combined NIR-PIT (p > 0.05, Tukey-Kramer test)
(FIG. 17D).
One of 9 mice in the combined NIR-PIT group achieved complete remission after
a single round of
NIR-PIT. Thus, combined CD25- and CD44-targeted NIR-PIT was superior
therapeutically to the
other two types of NIR-PIT in MOC1 tumors.
Example 15
Methods of Treating a Tumor
In one example, an antibody-IR700 molecule (such as anti-CD44-IR700) and an
immunomodulator (such as an anti-PD1 antibody, anti-PD-Li antibody, or anti-
CD25-IR700) are
administered to a subject with a tumor (day 1), such as a subject with cancer.
The subject is then
irradiated about 24 hours later with 50 J/cm2NIR light (day 2), and optionally
with 100 J/cm2 NIR
light 24 hours after the first irradiation (day 3). The immunomodulator is
also administered to the
subject on days 3, 5, and 7, at the same or a different (for example, lower)
dose.
The subject is monitored periodically for reduction of tumor size (such as
tumor weight or
volume), reduction in size or number of metastases, and/or survival (such as
overall survival,
progression-free survival, and/or disease-free survival).
In view of the many possible embodiments to which the principles of the
disclosure may be
applied, it should be recognized that illustrated embodiments are only
examples of the disclosure
and should not be considered a limitation on the scope of the invention.
Rather, the scope of the
invention is defined by the following claims. We therefore claim as our
invention all that comes
within the scope and spirit of these claims.
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